Methods for treating bacterial infections

ABSTRACT

The invention comprises methods for treating and preventing a bacterial infection in a subject, methods for preparing a medicament for use in treating and preventing a bacterial infection in a subject, and pharmaceutical and veterinary antibacterial compositions when used therein.

TECHNICAL FIELD

This invention relates to methods of treating and preventing a bacterialinfection in a subject, methods for preparing a medicament for use intreating and preventing a bacterial infection in a subject, andpharmaceutical and veterinary antibacterial compositions when usedtherein.

BACKGROUND ART

A marked increase in prevalence of multi-drug resistance indisease-causing Gram-positive (G+ve) (Staphylococcus aureus,Enterococcus spp. and Streptococcus pneumoniae) and Gram negative (G−ve)pathogens (Escherichia Enterobacter spp., Salmonella spp., Acinetobacterbaumannii, Klebsiella pneumoniae and Pseudomonas aeruginosa) hascoincided with an unprecedented global decline in investment in newanti-infective drugs. There are few currently registered alternativesfor multidrug resistant (MDR) bacterial infections, forcing cliniciansto consider older generation drugs such as colistin with narrow spectrumand considerable potential for toxic side-effects. In addition, thereare fewer novel classes of antiinfective therapeutics moving through thedrug development pipeline.

Since the year 2000, a period of almost 15 years, only 5 novel mode ofaction (MOA) antibacterial agents have been approved by the USFDA—linezolid (an oxazolidinone) in 2000, daptomycin (a lipopeptide) in2003, retapamulin (a pleuromutilin) in 2007, fidaxomicin (a macrolidetiacumicin) in 2011, and bedaquiline (a diarylquinoline) in 2012.Notably, none of these agents has significant activity against gramnegative bacteria. No novel MOA antibacterial agents were approved in2013 and to date in 2014 only tedizolid and dalbavancin, both analogs ofexisting classes, have been recommended for approval in the US. Whilethere are more than 300 anti-infective medicines in various stages ofdevelopment, the large majority of these medicines are previouslyapproved antibacterial compounds or their derivatives that areundergoing studies for new indications.

Furthermore, the prevalence of multidrug-resistance in animal-specificpathogens together with greater regulation of the registration and usageof antimicrobials in animals, has caused veterinarians to becomeincreasingly reliant on the traditional classes of antimicrobial agents.The risk of transfer of MDR zoonotic organisms from animals to humanshas also led to calls for further restrictions on the usage of somerecently registered antibacterial drugs such as the fluoroquinolones andthe third and fourth generation cephalosporins.

Epidemiology of Antibacterial Resistance Development in Pathogens ofHumans and Animals

Much of the evolution in resistance development is driven by changes inthe epidemiology of key MDR organisms. Once only restricted to humanhospitals and aged care facilities, methicillin resistant Staphylococcusaureus (MRSA) strains are now being isolated from the community inalarming proportions. Furthermore, community-acquired MRSA strains aremore likely to carry the Panton-Valentine leukocidin (PVL) toxin, avirulence factor linked to skin and soft tissue lesions as well as arapid, fulminating, necrotizing pneumonia with significant associatedmortality. Recently MRSA strains have become host-adapted in several keyanimal species including livestock, horses and companion animals andregular cases of human-to-animal and animal-to-human transfer are beingdocumented. This has important consequences for strain transmission andpublic health. A recent survey of 751 Australian veterinarians for MRSAnasal carriage found that a remarkable 21.4% of equine veterinarianswere MRSA-positive compared to 4.9% of small animal veterinarians and0.9% of veterinarians with little animal contact. These ecologicalshifts of MRSA together with the emergence of resistance to new drugsdeveloped specifically for MRSA such as linezolid confirm that new MRSAanti-infectives are urgently needed. Furthermore, hospitals that usevancomycin for treating MRSA then have to contend with outbreaks ofvancomycin-resistant enterococci (VRE) infections in their patients,once again with limited alternative antimicrobial choices.

The global emergence and spread within the community of highly virulentMDR Gram-negative (G−ve) bacteria such as E. coli O25b:ST131 confirmsthat bacterial pathogens can simultaneously evolve both virulence andresistance determinants. Echoing recent MRSA epidemiology, E. coliO25b:ST131, a major cause of urinary tract and bloodstream infections inhumans, has now been isolated from extraintestinal infections incompanion animals, and poultry. The increasing significance of E. coliO25b:ST131 and other MDR Enterobacteriaceae with combined resistance tofluoroquinolones and extended spectrum beta-lactams and carbapenems isanother worrying trend, especially considering there have been fewrecent breakthroughs in the development of G−ve spectrum anti-infectivesapart from incremental advances in the carbapenem family.

The World Health Organisation has identified antibiotic resistance asone of the three major future threats to global health. A recent reportfrom the US Centers for Disease Control and Prevention (CDC) estimatedthat “in the United States, more than two million people are sickenedevery year with antibiotic-resistant infections, with at least 23,000dying as a result.” The extra medical costs, in the USA alone,associated with treating and managing a single case ofantibiotic-resistant infection are estimated to be between US$18,588 andUS$29,069 per year resulting in an overall direct cost to the US healthsystem of over US$20 billion annually. In addition, the cost to UShouseholds in terms of lost productivity is estimated at over US$35billion per annum. Twenty five thousand patients in the European Union(EU) still die annually from infection with MDR bacteria despite many EUcountries having world's best practice hospital surveillance andinfection control strategies. The EU costs from health care expenses andlost productivity associated with MDR infections are estimated to be atleast

1.5 billion per year.

There is an unmet clinical need for antibacterial agents with novelmechanisms of action to supplement and replace currently availableantibacterial agents, the efficacy of which is increasingly underminedby antibacterial resistance mechanisms. There remains a need foralternative antibacterials in the treatment of infection bymulti-resistant bacteria. However, as reported by the InfectiousDiseases Society of America and the European Centre for Disease Controland Prevention, few new drugs are being developed that offer promisingresults over existing treatments (Infectious Diseases Society of America2010, Clinical Infectious Diseases, 50(8):1081-1083).

It is an object of the present invention to overcome at least one of thefailings of the prior art.

The discussion of the background art set out above is intended tofacilitate an understanding of the present invention only. Thediscussion is not an acknowledgement or admission that any of thematerial referred to is or was part of the common general knowledge asat the priority date of the application.

SUMMARY OF INVENTION

According to one aspect of the invention, there is provided a method oftreating or preventing a bacterial colonisation or infection in asubject, the method comprising the step of: administering atherapeutically effective amount of robenidine, or a therapeuticallyacceptable salt thereof, to the subject. In this aspect, the bacterialcolonisation or infection is caused by a bacterial agent.

According to another aspect of the invention, there is provided the useof robenidine, or a therapeutically acceptable salt thereof, in themanufacture of medicament for the treatment of a bacterial colonisationor infection in a subject. In this aspect, the bacterial colonisation orinfection is caused by a bacterial agent.

The subject may be any subject capable of colonisation or infection bybacteria. The subject may be mammalian, or may be piscine or avian.Preferably, the subject is selected from the group comprising, but notlimited to, human, canine, feline, bovine, ovine, caprine, otherruminant species, porcine, equine, avian, or piscine.

As used herein, the term robenidine, (also known as1,2-bis[(E)-(4-chlorophenyl)methylideneamino]guanidine, or, as describedby this specification, NCL812) refers to a compound having the followingchemical structure:

The robenidine may be administered to the subject in a dose selectedfrom the group comprising 0.1 mg/kg to 250 mg/kg body weight, preferably1 mg/kg to 100 mg/kg body weight, and more preferably 5 mg/kg to 50mg/kg body weight. The robenidine may be administered to the subjectusing a dosing schedule selected from the group consisting of: hourly, 3times daily; twice daily; daily; every second day; twice weekly; onceweekly; once fortnightly; once monthly; once every two months or byconstant rate or variable rate infusion. Preferably, the robenidine isadministered until colonisation or the signs and symptoms of infectionhave at least been partially treated or alleviated.

In one embodiment, the concentration of robenidine (or a robenidinemetabolite) in the subject's blood after treatment is within a rangeselected from the group comprising, but not limited to: between 0.1 and10 ug/mL at 2 hours, 1 and 200 ug/mL after 12 hours; between 0.1 and 5ug/mL after 24 hrs; between 0.01 and 2 ug/mL after 48 hours; between0.0001 and 1 ug/mL after 72 hrs. Preferably, the concentration isselected from the group comprising, but not limited to: less than 200ug/mL after 12 hours; less than 5 ug/mL after 24 hours; less than 1 ug/Lafter 48 hours and less than 0.5 ug/mL after 72 hours.

The agent causing the bacterial infection is a bacterial agent. In onepreferred embodiment, the agent is not a protozoan species. In onepreferred embodiment, the agent is not a coccidian protozoan. Morepreferably, the agent is not Clostridium perfringens nor a heterotrophicbacterial species present in soil samples collected by Hansen et al fromJyndevad Denmark as discussed in the following papers: Hansen et al.2012, Chemosphere, 86:212-215; and Hansen et al. 2009, EnvironmentalPollution 157:474-480.

In another embodiment, the bacterial agent is gram negative. In anotherembodiment, the bacterial agent is gram positive. In another embodiment,the bacterial agent has no cell wall. In another embodiment, thebacterial infection is caused by a mixture of at least two agentsselected from the group consisting of: gram negative, gram positive andbacterial agents with no cell wall.

The bacterial agent causing the bacterial infection may be a grampositive bacterial agent selected from the group comprising, but notlimited to, Staphylococcus spp, Streptococci, Enterococcus spp,Leuconostoc spp, Corynebacterium spp, Arcanobacteria spp, Trueperellaspp, Rhodococcus spp, Bacillus spp, Anaerobic Cocci, AnaerobicGram-Positive Nonsporulating Bacilli, Actinomyces spp, Clostridium spp,Nocardia spp, Erysipelothrix spp, Listeria spp, Kytococcus spp,Mycoplasma spp, Ureaplasma spp, and Mycobacterium spp.

In one embodiment, the bacterial agent is gram positive and selectedfrom the group comprising, but not limited to Staphylococcus spp.Examples of Staphylococcus spp include Staphylococcus epidermidis,Staphylococcus haemolyticus, Staphylococcus lugdunensis, Staphylococcussaprophyticus, Staphylococcus auricularis, Staphylococcus capitis,Staphylococcus caprae, Staphylococcus carnosus, Staphylococcus cohnii,Staphylococcus hominis, Staphylococcus pasteuri, Staphylococcuspettenkoferi, Staphylococcus pulvereri, Staphylococcus saccharolyticus,Staphylococcus simulans, Staphylococcus schleiferi, Staphylococcuswarneri, Staphylococcus xylosus, Staphylococcus arlettae, Staphylococcuscaseolyticus, Staphylococcus chromogenes, Staphylococcus condimenti,Staphylococcus delphini, Staphylococcus equorum, Staphylococcus felis,Staphylococcus fleurettii, Staphylococcus gallinarum, Staphylococcushyicus, Staphylococcus intermedius, Staphylococcus kloosii,Staphylococcus lentus, Staphylococcus lutrae, Staphylococcus muscae,Staphylococcus nepalensis, Staphylococcus piscifermentans,Staphylococcus pseudintermedius, Staphylococcus sciuri, Staphylococcussimiae, Staphylococcus succinus, and Staphylococcus vitulinus.

In another embodiment, the bacterial agent is gram positive and isselected from the group comprising, but not limited to, Streptococcusspp. Examples of Streptococcus spp include Streptococcus agalactiae,Streptococcus alactolyticus, Streptococcus anginosus, Streptococcuscanis, Streptococcus constellatus, Streptococcus cricetus, Streptococcuscristatus, Streptococcus downei, Streptococcus dysgalactiae subsp.dysgalactiae, Streptococcus dysgalactiae subsp. equisimilis,Streptococcus equi subsp. equi, Streptococcus equi subsp. zooepidemicus,Streptococcus ferus, Streptococcus gallolyticus subsp. gallolyticus(formerly Streptococcus bovis biotype i), Streptococcus gallolyticussubsp. pasteurianus (formerly Streptococcus bovis biotype ii/2),Streptococcus gordonii, Streptococcus hyointestinalis, Streptococcushyovaginalis, Streptococcus infantarius, Streptococcus infantarius subspinfantarius, Streptococcus infantis, Streptococcus iniae, Streptococcusintermedius, Streptococcus lutetiensis (formerly Streptococcus bovisbiotype ii.1), Streptococcus macaccae, Streptococcus mitis,Streptococcus mutans, Streptococcus oralis, Streptococcus orisratti,Streptococcus parasanguinis, Streptococcus peroris, Streptococcuspneumoniae, Streptococcus porcinus, Streptococcus pseudintermedius,Streptococcus pyogenes, Streptococcus ratti, Streptococcus salivarius,Streptococcus sanguinis, Streptococcus sobrinus, Streptococcus suis,Streptococcus thermophilus, Streptococcus vestibularis, andNutritionally Variant (Deficient) Streptococci (Abiotrophia defectiva,Granulicatella adiacens, Granulicatella elegans, and Granulicatellapara-adiacens) and related species such as Rothia mucilaginosa (formerlyStomatococcus mucilaginosus) and Pediococcus.

In another embodiment, the bacterial agent is gram positive and selectedfrom the group comprising, but not limited to, Enterococcus spp.Examples of Enterococcus spp include Enterococcus faecalis, Enterococcusfaecium, Enterococcus gallinarum, Enterococcus durans, Enterococcusavium, Enterococcus raffinosus, Enterococcus pallens, Enterococcusgilvus, Enterococcus cecorum, Enterococcus malodoratus, Enterococcusitalicus, Enterococcus sanguinicola, Enterococcus mundtii, Enterococcuscasseliflavus/flavescens, Enterococcus dispar, Enterococcus hirae,Enterococcus pseudoavium, and Enterococcus bovis.

In another embodiment, the bacterial agent is gram positive and selectedfrom the group comprising, but not limited to, Leuconostoc spp. Examplesof Leuconostoc spp include Leuconostoc mesenteroides, Leuconostocpseudomesenteroides, Leuconostoc paramesenteroides, Leuconostoc citreum,and Leuconostoc lactis.

In another embodiment, the bacterial agent is gram positive and selectedfrom the group comprising, but not limited to, Corynebacterium spp.Examples of Corynebacterium spp include nonlipophilic, fermentativeCorynebacterium spp such as Corynebacterium ulcerans, Corynebacteriumpseudotuberculosis, Corynebacterium xerosis, Corynebacterium striatum,Corynebacterium minutissimum, Corynebacterium amycolatum,Corynebacterium glucuronolyticum, Corynebacterium argentoratense,Corynebacterium matruchotii, Corynebacterium riegelii, Corynebacteriumconfusum, Corynebacterium cystidis, Corynebacterium diphtheria,Corynebacterium simulans, Corynebacterium sundvallense, Corynebacteriumthomssensii, Corynebacterium freneyi, and Corynebacterium aurimucosum,nonlipophilic, nonfermentative Corynebacterium spp such asCorynebacterium afermentans afermentans, Corynebacterium auris,Corynebacterium pseudodiphtheriticum, and Corynebacterium propinquum andlipophilic Corynebacterium spp such as Corynebacterium jeikeium,Corynebacterium urealyticum, Corynebacterium afermentans lipophilum,Corynebacterium accolens, Corynebacterium macginleyi, Corynebacteriumtuberculostearum, Corynebacterium kroppenstedtii, Corynebacteriumkutscheri, Corynebacterium pilosum, Corynebacterium bovis, CDCcoryneform groups F-1 and G, and Corynebacterium lipophiloflavum, andother Corynebacterium spp such as Turicella, Arthrobacter,Brevibacterium, Dermabacter, Rothia, Oerskovia, Microbacterium, andLeifsonia aquatica.

In another embodiment, the bacterial agent is gram positive and selectedfrom the group comprising, but not limited to, Arcanobacteria spp.Examples of Arcanobacteria spp include A. haemolyticum, A. pyogenes (nowknown as Trueperella pyogenes, originally known as Actinomycespyogenes), and A. bernardiae.

In another embodiment, the bacterial agent is gram positive and selectedfrom the group comprising, but not limited to, Rhodococcus spp. Examplesof Rhodococcus spp include Rhodococcus equi, Rhodococcus erythropolis,Rhodococcus fasciens, and Rhodococcus rhodochrous.

In another embodiment, the bacterial agent is gram positive and selectedfrom the group comprising, but not limited to, Gordonia spp.

In another embodiment, the bacterial agent is gram positive and selectedfrom the group comprising, but not limited to, Tsukamurella spp.

In another embodiment, the bacterial agent is gram positive and selectedfrom the group comprising, but not limited to, Acholeplasma spp.

In another embodiment, the bacterial agent is gram positive and selectedfrom the group comprising, but not limited to, Actinobacteria such asCrossiella equi.

In another embodiment, the bacterial agent is gram positive and selectedfrom the group comprising, but not limited to, Bacillus spp. Examples ofBacillus spp include Bacillus anthracis, Bacillus cereus, Bacilluscirculans, Bacillus licheniformis, Bacillus megaterium, Bacilluspumilus, Bacillus sphaericus, Bacillus subtilis, Brevibacillus brevis,Brevibacillus laterosporus, and Paenibacillus alvei.

In another embodiment, the bacterial agent is gram positive and selectedfrom the group comprising, but not limited to, Anaerobic Cocci. Examplesof Anaerobic Cocci include Anaerococcus murdochii, Anaerococcusprevotii, Anaerococcus tetradius, Anaerococcus octavius, Anaerococcushydrogenalis, Anaerococcus lactolyticus, Anaerococcus vaginalis,Atopobium parvulum, Finegoldia magna, Gallicola barnesae, Gemellaasaccharolytica, Gemella bergeri, Gemella cuniculi, Gemella haemolysans,Gemella morbillorum, Gemella palaticanis, Gemella sanguinis; Parvimonasmicra, Peptococcus niger, Peptoniphilus asaccharolyticus, Peptoniphilusgorbachii, Peptoniphilus indolicus, Peptoniphilus harei, Peptoniphilusivorii, Peptoniphilus lacrimalis, Peptoniphilus olsenii,Peptostreptococcus stomatis, Peptostreptococcus anaerobius, Ruminococcusproductus, Slackia heliotrinireducens, and Staphylococcussaccharolyticus.

In another embodiment, the bacterial agent is gram positive and selectedfrom the group comprising, but not limited to, Anaerobic Gram-PositiveNonsporulating Bacilli. Examples of Anaerobic Gram-PositiveNonsporulating Bacilli include Alloscardovia omnicolens, Atopobiumspecies (such as Atopobium minutum, Atopobium rimae, Atopobium parvulum,and Atopobium vaginae), Bifidobacteria (such as Bifidobacteriaadolescentis, Bifidobacteria dentium, Bifidobacteria scardovii),Catabacter hongkongensis, Collinsella aerofaciens, Eggerthella (such asEggerthella lenta, Eggerthella hongkongensis and Eggerthella sinensis),Eubacterium and related species (such as Eubacterium nodatum,Eubacterium tenue, Eubacterium brachy, Eubacterium infirmum, Eubacteriumminutum, Eubacterium nodatum, Eubacterium saphenum, Eubacterium sulci,Filifactor alocis, Mogibacterium timidum, Mogibacterium vescum,Pseudoramibacter alactolyticus, Bulleidia extructa, and Solobacteriummoorei), Lactobacillus species (such as Lactobacillus rhamnosus,Lactobacillus casei, Lactobacillus fermentum, Lactobacillus gasseri,Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus inersand Lactobacillus ultunensis), Mobiluncus species (such as Mobiluncuscurtisii, Mobiluncus mulieris), Moryella indoligenes, Olsenella oralspecies (such as Olsenella uli and Olsenella profuse), Oribacteriumsinus, Propionibacterium (such as Propionibacterium acnes andPropionibacterium propionicum), Slackia exigua, and Turicibactersanguine.

In another embodiment, the bacterial agent is gram positive and selectedfrom the group comprising, but not limited to, Actinomyces spp. Examplesof Actinomyces spp include Actinomyces israelii, Actinomyces naeslundii,Actinomyces viscosus, Actinomyces odontolyticus, Actinomyces meyeri, andActinomyces gerencseriae (formerly Actinomyces israelii serotype II),Actinomyces europaeus, Actinomyces neuii, Actinomyces radingae,Actinomyces graevenitzii, Actinomyces hordeovulneris, Actinomycesturicensis, Actinomyces georgiae, Arcanobacterium (Actinomyces)pyogenes, Arcanobacterium (Actinomyces) bernardiae, Actinomyces funkei,Actinomyces lingnae, Actinomyces houstonensis, and Actinomycescardiffensis.

In another embodiment, the bacterial agent is gram positive and selectedfrom the group comprising, but not limited to, Clostridium spp. Examplesof Clostridium spp include Clostridium baratii, Clostridiumbifermentans, Clostridium botulinum, Clostridium botulinum (types A, B,C, D, E, F, G), Clostridium butyricum, Clostridium difficile,Clostridium histolyticum, Clostridium novyi (type A), Clostridium novyi(type B), Clostridium perfringens, Clostridium perfringens (types A-E),Clostridium ramosum, Clostridium septicum, Clostridium sordelli,Clostridium sphenoides, Clostridium tedium, and Clostridium tetani.

In another embodiment, the bacterial agent is gram positive and selectedfrom the group comprising, but not limited to, Nocardia spp. Examples ofNocardia spp include Nocardia asteroides, Nocardia brasiliensis,Nocardia farcinica, Nocardia nova, Nocardia otitidiscaviarum, andNocardia transvalensis.

In another embodiment, the bacterial agent is gram positive and selectedfrom the group comprising, but not limited to, Erysipelothrix spp, suchas Erysipelothrix rhusiopathiae.

In another embodiment, the bacterial agent is gram positive and selectedfrom the group comprising, but not limited to, Listeria spp, such asListeria monocytogenes.

In another embodiment, the bacterial agent is gram positive and selectedfrom the group comprising, but not limited to, Kytococcus spp, such asKytococcus schroeteri.

In another embodiment, the bacterial agent is gram positive and selectedfrom the group comprising, but not limited to, Mycobacterium spp.Examples of Mycobacterium spp include Mycobacterium abscessus,Mycobacterium arupense, Mycobacterium asiaticum, Mycobacteriumaubagnense, Mycobacterium avium complex, Mycobacterium bolletii,Mycobacterium bolletii, Mycobacterium branderi, Mycobacterium canettii,Mycobacterium caprae, Mycobacterium celatum, Mycobacterium chelonae,Mycobacterium chimaera, Mycobacterium colombiense, Mycobacteriumconceptionense, Mycobacterium conspicuum, Mycobacterium elephantis,Mycobacterium farcinogenes, Mycobacterium florentinum, Mycobacteriumfortuitum group, Mycobacterium genavense, Mycobacterium goodii,Mycobacterium haemophilum, Mycobacterium heckeshornense, Mycobacteriumheidelbergense, Mycobacterium houstonense, Mycobacterium immunogenum,Mycobacterium interjectum, Mycobacterium intracellulare, Mycobacteriumsenegalense, Mycobacterium africanum, Mycobacterium avium subspparatuberculosis, Mycobacterium kansasii, Mycobacterium lacus,Mycobacterium lentiflavum, Mycobacterium leprae, Mycobacteriumlepraemurium, Mycobacterium mageritense, Mycobacterium malmoense,Mycobacterium marinum, Mycobacterium massiliense, Mycobacterium microti,Mycobacterium montefiorense (eels), Mycobacterium moracense,Mycobacterium mucogenicum, Mycobacterium nebraskense, Mycobacteriumneoaurum, Mycobacterium novocastrense, Mycobacterium palustre,Mycobacterium parmense, Mycobacterium phlei, Mycobacterium phocaicum,Mycobacterium pinnipedii, Mycobacterium porcinum, Mycobacteriumpseudoshottsii (fish), Mycobacterium pseudotuberculosis, Mycobacteriumsaskatchewanense, Mycobacterium scrofulaceum, Mycobacterium senuense,Mycobacterium septicum, Mycobacterium simiae, Mycobacterium smegmatis,Mycobacterium szulgai, Mycobacterium terrae/chromogenicum complex,Mycobacterium triplex, Mycobacterium tuberculosis, Mycobacteriumtusciae, Mycobacterium ulcerans, Mycobacterium wolinskyi, andMycobacterium xenopi.

In another embodiment, the bacterial agent is gram positive and selectedfrom the group comprising, but not limited to, Trueperella spp. Examplesof Trueperella spp include Trueperella abortisuis, Trueperellabernardiae, Trueperella bialowiezensis, Trueperella bonasi, Trueperellapyogenes (Arcanobacterium pyogenes).

In another embodiment, the bacterial agent is gram positive, gramnegative or does not have a cell wall and selected from the groupcomprising, but not limited to, livestock pathogens. Examples oflivestock pathogens include Actinobaculum suis, Actinomyces bovis,Arcanobacterium pyogenes, Bacillus anthracis, cereus, licheniformis,pumilus, melaninogenicus, subtilis, Clostridium botulinum, chauvoei,haemolyticum, novyi, perfringens, septicum, sordellii, tetani, colinum,Corynebacterium pseudotuberculosis, renale, Dermatophilus congolensis,Enterococcus spp (such as E. faecalis, E. faecium, E. durans, E. avium,E. hirae), Erysipelothrix rhusiopathiae, Listeria ivanovii, grayi,innocua, seeligeri, welshimeri, monocytogenes, Mycobacterium avium,bovis, paratuberculosis (Johne's Disease), Mycoplasma (such ascapricolum subsp. capripneumoniae, subsp. capricolum, M. mycoides subspmycoides, M. agalactiae, M. ovipneumoniae, M. conjunctivae, M. arginini,M. bovis, and M. putrefaciens) Mycoplasma bovis, dispar, mycoides subsp.mycoides (such as Contagious bovine pleuropneumonia CBPP) Mycoplasmagallisepticum (MG), iowae meleagridis (MM), synoviae (MS) Mycoplasmahaemosuis (formerly Eperythrozoon suis), alkalescens, bovigenitalum,bovirhinis, bovoculi, californicum, canadense, cynos, equigenitalium,gateae, haemocanis, haemofelis, hyopneumoniae, hyorhinis, hyosynoviae,iowae, leachii, meleagridis, mycoides subsp capri, wenyonii, suis,Rhodococcus equi, Staphylococcus epidermidis, Staphylococcus simulans,Staphylococcus felis, Staphylococcus xylosus, Staphylococcuschromogenes, Staphylococcus warneri, Staphylococcus haemolyticus,Staphylococcus sciuri, Staphylococcus saprophyticus, Staphylococcushominis, Staphylococcus caprae, Staphylococcus cohnii subsp. cohnii,Staphylococcus cohnii subsp. urealyticus, Staphylococcus capitis subsp.capitis, Staphylococcus capitis subsp. urealyticus, Staphylococcushyicus, Staphylococcus aureus, Staphylococcus pseudintermedius,Staphylococcus delphini, Staphylococcus schleiferi subsp. coagulans,Staphylococcus aureus subsp. anaerobius, Streptococcus uberis,Streptococcus canis, Streptococcus agalactiae, Streptococcusdysgalactiae, Streptococcus pyogenes, Streptococcus bovis, Streptococcusequi subsp. Zooepidemicus, Streptococcus equinus, Streptococcus equi(Streptococcus equi subsp equi), Streptococcus equisimilis(Streptococcus dysgalactiae subsp equisimilis), porcinus, suis,zooepidemicus, Streptococcus zooepidemicus (Streptococcus equi subspzooepidemicus), Streptococcus dysgalactiae subsp. equisimilis,Propionibacterium acnes, Propionibacterium granulosum, Eubacterium,Peptococcus indolicus, and Peptostreptococcus anaerobius; and variousspecies of the following Gram negative genera: Actinobacillus,Aeromonas, Anaplasma, Arcobacter, Avibacterium, Bacteroides, Bartonella,Bordetella, Borrelia, Brachyspira, Brucella, Campylobacter,Capnocytophaga, Chlamydia, Chlamydophila, Chryseobacterium, Coxiella,Cytophaga, Dichelobacter, Edwardsiella, Ehrlichia, Escherichia,Flavobacterium, Francisella, Fusobacterium, Gallibacterium, Haemophilus,Histophilus, Klebsiella, Lawsonia, Leptospira, Mannheimia, Megasphaera,Moraxella, Neorickettsia, Nicoletella, Ornithobacterium, Pasteurella,Photobacterium, Piscichlamydia, Piscirickettsia, Porphyromonas,Prevotella, Proteus, Pseudomonas, Rickettsia, Riemerella, Salmonella,Streptobacillus, Tenacibaculum, Vibrio, and Yersinia.

In another embodiment, the bacterial agent is gram positive and selectedfrom the group comprising, but not limited to, pathogens of companionanimal species such as cats, dogs and horses. Examples of such pathogensinclude equine pathogens such as Streptococcus equi, Streptococcuszooepidemicus, Rhodococcus equi, Clostridium difficile, Clostridiumperfringens, Corynebacterium pseudotuberculosis, Clostridium piliforme,Actinomyces bovis, Staphylococcus aureus, beta haemolytic Steptococcusspp, Dermatophilus congolense, Clostridiium tetani, and Clostridiumbotulinum. Further examples include pathogens of dogs and cats such asStaphylococcus spp, Streptococcus spp, Clostridium spp, Actinomyces spp,Enterococcus spp, Nocardia spp, Mycoplasma spp, and Mycobacterium spp.

In another embodiment, the bacterial agent is gram negative and selectedfrom the group consisting of the following representative families andspecies: Acetobacteraceae:—Roseomonas cervicalis; Roseomonas fauriae;Roseomonas gilardii.—Aeromonadaceae:—Aeromonas allosaccharophila;Aeromonas aquariorum; Aeromonas caviae; Aeromonas hydrophila (andsubspecies); Aeromonas salmonicida; Aeromonas shubertii; Aeromonasveronii biovar sobria (Aeromonas sobria).—Alcaligenaceae:—Achromobacterxylosoxidans; Alcaligenes faecalis; Bordetella ansorpii; Bordetellaavium; Bordetella bronchiseptica; Bordetella Bordetella holmesii;Bordetella parapertussis; Bordetella pertussis; Bordetella petrii;Bordetella trematum; Oligella ureolytica; Oligellaurethralis.—Anaplasmataceae:—Anaplasma phagocytophilum; Anaplasmaplatys; Anaplasma bovis; Anaplasma centrale; Anaplasma marginale;Anaplasma odocoilei; Anaplasma ovis; Ehrlichia canis; Ehrlichiachaffeensis; Ehrlichia ewingii; Ehrlichia muris; Ehrlichia ovina;Ehrlichia ruminantium; Neoehrlichia lotoris; Neoehrlichia mikurensis;Neorickettsia helminthoeca; Neorickettsia risticii, Neorickettsiasennetsu; Wolbachia pipientis.—Armatimonadaceae:—Armatimonasrosea.—Bacteroidaceae:—Bacteroides forsythus; Bacteroides fragilis;Bacteroides melaninogenicus; Bacteroides ruber; Bacteroidesurealtyicus.—Bartonellaceae:—Bartonella alsatica; Bartonella australis;Bartonella bacilliformis; Bartonella birtlesii; Bartonella bovis;Bartonella capreoli; Bartonella chomelii; Bartonella clarridgeiae;Bartonella doshiae; Bartonella elizabethae; Bartonella grahamii;Bartonella henselae; Bartonella koehlerae; Bartonella peromysci;Bartonella phoceensis; Bartonella quintana; Bartonellarattimassiliensis; Bartonella rochalimae; Bartonella schoenbuchensis;Bartonella talpae; Bartonella tamiae; Bartonella taylorii; Bartonellatribocorum; Bartonella vinsonii subsp berkhoffii; Bartonella vinsoniisubsp. arupensis; Bartonella vinsonii subsp.vinsonii.—Bdellovibrionaceae:—Bdellovibriospp.—Brachyspiraceae:—Brachyspira spp including Brachyspira hampsonii,Brachyspira hyodysenteriae, Brachyspira murdochii, Brachyspirapilosicoli.—Brucellaceae:—Brucella abortus; Brucella canis; Brucellaceti; Brucella melitensis; Brucella ovis; Brucella pinnipedialis;Brucella suis; Ochrobactrum anthropi; Ochrobactrumintermedium.—Burkholderiaceae:—Burkholderia aboris; Burkholderiaambifaria (genomovar VII); Burkholderia anthina (genomovar VIII);Burkholderia cenocepacia (genomovar III); Burkholderia cepacia(genomovar I); Burkholderia diffusa; Burkholderia dolosa (genomovar VI);Burkholderia latens; Burkholderia mallei; Burkholderia metallica;Burkholderia multivorans (genomovar II); Burkholderia pseudomallei;Burkholderia pyrrocinia (genomovar IX); Burkholderia seminalis;Burkholderia stabilis (genomovar IV); Burkholderia ubonensis (genomovarX); Burkholderia vietnamiensis (genomovar V); Cupriavidus pauculus;Cupriavidus gilardii; Ralstonia pickettii; Ralstonia mannitolilytica;Sphaerotilus hippei; Sphaerotilus montanus; Sphaerotilusnatans.—Campylobacteraceae:—Arcobacter spp including Arcobacterskirrowii; Campylobacter coli; Campylobacter concisus; Campylobactercurvus; Campylobacter fetus; Campylobacter gracilis; Campylobacterhelveticus; Campylobacter hominis; Campylobacter hyointestinalis;Campylobacter insulaenigrae; Campylobacter jejuni; Campylobacterlanienae; Campylobacter lari; Campylobacter laridis; Campylobactermucosalis; Campylobacter rectus; Campylobacter showae; Campylobactersputorum; Campylobacter upsaliensis.—Candidatus:—Piscichlamydiasalmonis.—Cardiobacteriaceae:—Cardiobacterium hominis; Cardiobacteriumvalvarum; Dichelobacter nodosus.—Chlamydiaceae:—Chlamydia spp includingChlamydia avium, Chlamydia gallinacea, Chlamydia muridarum, Chlamydiasuis, Chlamydia trachomatis; Chlamydophila spp including Chlamydophilapneumoniae, Chlamydophila pecorum, Chlamydophila psittaci, Chlamydophilaabortus, Chlamydophila caviae, and Chlamydophilafelis.—Chthonomonadaceae:—Chthonomonascalidirosea.—Comamonadaceae:—Comamonas testosteroni; Verminephrobacterspp.—Coxiefiaceae:—Coxiella burnetii.—Cytophagaceae:—Cytophagacolumnaris; Cytophaga hutchinsonii; Flexibacter echinicida; Flexibacterelegans; Flexibacter flexilis; Flexibacter litoralis; Flexibacterpolymorphus; Flexibacter roseolus; Flexibacterruber.—Desulfovibrionaceae:—Bilophila wadsworthia; Lawsoniaintracellularis.—Enterobacteriaceae:—Cedecea davisae; Cedecea lapagei;Cedecea neteri; amalonaticus; Citrobacter diversus; Citrobacterfreundii; Citrobacter koseri; Cronobacter condimenti; Cronobacterdublinensis; Cronobacter helveticus; Cronobacter malonaticus;Cronobacter muytjensii; Cronobacter pulveris; Cronobacter sakazakii;Cronobacter turicensis; Cronobacter universalis; Cronobacterzurichensis; Edwardsiella ictaluri; Edwardsiella tarda; Enterobacteraerogenes; Enterobacter agglomerans; Enterobacter cloacae; Enterobactercowanii; Escherichia albertii; Escherichia coli, including AIEC=adherentinvasive E. coli, EaggEC=enteroaggregative E. coli;EHEC=enterohemorrhagic E. coli; EIEC=enteroinvasive E. coli;EPEC=enteropathogenic E. coli; ETEC=enterotoxigenic E. coli;ExPEC=extraintestinal pathogenic E. coli, NMEC=neonatal meningitis E.coli, NTEC=necrotoxigenic E. coli, UPEC=uropathogenic E. coli;Escherichia fergusonii; Ewingella americana; Hafnia alvei; Hafniaparalvei; Klebsiella granulomatis; Klebsiella oxytoca; Klebsiellapneumoniae; Kluyvera ascorbata; Kluyvera cryocrescens; Morganellamorganii; Pantoea (formerly Enterobacter) agglomerans; Photorhabdusasymbiotica; Plesiomonas shigelloides; Proteus mirabilis; Proteuspenneri; Proteus vulgaris; Providencia alcalifaciens; Providenciarettgeri; Providencia stuartii; Raoultella electrica; Raoultellaornithinolytica; Raoultella planticola; Raoultella terrigena; Salmonellabongori, Salmonella enterica subspecies enterica (many serotypes);Serratia liquifaciens; Serratia marcesans; Shigella boydii; Shigelladysenteriae; Shigella flexneri; Shigella sonnei; Yersiniaenterocolitica; Yersinia pestis; Yersinia pseudotuberculosis; Yersiniaruckeri.—Fimbriimonadaceae:—Fimbriimonasginsengisoli.—Flavobacteriaceae:—Bergeyella zoohelcum; Capnocytophagacanimorsus; Capnocytophaga cynodegmi; Capnocytophaga gingivalis;Capnocytophaga granulosa; Capnocytophaga haemolytica; Capnocytophagaleadbetteri; Capnocytophaga ochracea; Capnocytophaga sputigena;Chryseobacterium indologenes; Chryseobacterium piscicola;Elizabethkingia meningoseptica; Flavobacterium branchiophilum;Flavobacterium columnare; Flavobacterium oncorhynchi; Flavobacteriumpiscicida; Flavobacterium psychrophilum; Myroides odoratus; Myroidesodoratimimus; Ornithobacterium rhinotracheale; Riemerella anatipestifer;Riemerella columbina; Riemerella columbipharyngis; Tenacibaculumdicentrarchi; Tenacibaculum discolour; Tenacibaculum gallaicum;Tenacibaculum maritimum; Tenacibaculum soleae; Weeksellavirosa.—Francisellaceae:—Francisella tularensis subsp. tularensis;Francisella tularensis subsp. holarctica; Francisella tularensis subsp.novicida; Francisella philomiragia; Francisella noatunensis; Francisellanoatunensis subsp. orientalis (also termed Francisellaasiatica).—Fusobacteriaceae:—Fusobacterium spp. including Fusobacteriumnecrophorum, Fusobacterium nucleatum, Fuso-bacteriumpolymorphum.—Helicobacteraceae:—Helicobacter cinaedi; Helicobacterfenneffiae; Helicobacter pylori.—Legionellaceae:—Legionella pneumophilaand other species including; Legionella anisa; Legionellabirminghamensis; Legionella bozemannii; Legionella cincinnatiensis;Legionella dumoffii; Legionella feeleii; Legionella gormanii; Legionellahackeliae; Legionella jordanis; Legionella lansingensis; Legionellalongbeachae; Legionella maceachemii; Legionella micdadei; Legionellaoakridgensis; Legionella parisiensis; Legionella sainthelens; Legionellatusconensis; Legionella wadsworthii; Legionellawaltersii.—Leptospiraceae:—Leptospira alexanderi (including Leptospiraalexanderi serovar Hebdomadis, Leptospira alexanderi serovar Manhao 3);Leptospira alstoni (including Leptospira alstoni serovar Pingchang,Leptospira alstoni serovar Sichuan); Leptospira biflexa (includingLeptospira biflexa serovar Ancona, Leptospira biflexa serovar Canela);Leptospira borgpetersenii (including Leptospira borgpetersenii serovarHardjo, Leptospira borgpetersenii serovar Hardjo-bovis, Leptospiraborgpetersenii serovar Pomona, Leptospira borgpetersenii serovarTarassovi); Leptospira broomii (including Leptospira broomii serovarHurstbridge); Leptospira fainei (including Leptospira fainei serovarHurstbridge); Leptospira idonii; Leptospira inadai (including Leptospirainadai serovar Lyme, Leptospira inadai serovar Malaya); Leptospirainterrogans (including Leptospira interrogans serovar Australis,Leptospira interrogans serovar Autumnalis, Leptospira interrogansserovar Bratislava, Leptospira interrogans serovar Canicola, Leptospirainterrogans serovar Grippotyphosa, Leptospira interrogans serovarHardjo, Leptospira interrogans serovar Hardjo-bovis, Leptospirainterrogans serovar Icterohaemorrhagiae, Leptospira interrogans serovarPomona, Leptospira interrogans serovar Pyrogenes, Leptospira interrogansserovar Tarassovi); Leptospira kirschneri (including Leptospirakirschneri serovar Bulgarica, Leptospira kirschneri serovar Cynopteri,Leptospira kirschneri serovar Grippotyphosa); Leptospira kmetyi;Leptospira licerasiae; Leptospira meyeri (including Leptospira meyeriserovar Sofia); Leptospira noguchii (including Leptospira noguchiiserovar Panama, Leptospira noguchii serovar Pomona); Leptospirasantarosai; Leptospira terpstrae; Leptospira vanthielii; Leptospiraweilii (including Leptospira weilii serovar Celledoni, Leptospira weiliiserovar Sarmin); Leptospira wolbachii; Leptospira wolffii; Leptospirayanagawae.—Leptotrichiaceae:—Leptotrichia buccalis; Streptobacillusmoniliformis.—Methylobacteriaceae:—Methylobacterium extorquens group;Methylobacterium fujisawaense; Methylobacterium mesophilicum;Methylobacterium zatmanii.—Moraxellaceae:—Acinetobacter baumannii(genomic species 2); Acinetobacter baylyi; Acinetobacter bouvetii;Acinetobacter calcoaceticus (genomic species 1); Acinetobacter gerneri;Acinetobacter grimontii; Acinetobacter haemolyticus (genomic species 4);Acinetobacter johnsonii (genomic species 7); Acinetobacter junii(genomic species 5); Acinetobacter lwoffi (genomic species 8/9);Acinetobacter parvus; Acinetobacter radioresistens (genomic species 12);Acinetobacter schindleri; Acinetobacter tandoii; Acinetobactertjernbergiae; Acinetobacter towneri; Acinetobacter ursingii;Acinetobacter venetianus; Moraxella atlantae; Moraxella boevrei;Moraxella bovis; Moraxella bovoculi; Moraxella canis; Moraxella caprae;Moraxella catarrhalis; Moraxella caviae; Moraxella cuniculi; Moraxellaequi; Moraxella lacunata; Moraxella lincolnii; Moraxella macacae;Moraxella nonliquefaciens; Moraxella oblonga; Moraxella osloensis;Moraxella ovis; Moraxella phenylpyruvica; Moraxella pluranimalium;Moraxella porci.—Moritefiaceae: —Moritella abyssi; Moritella dasanensis;Moritella japonica; Moritella marina; Moritella pro-funda; Moritellaviscosa; Moritella yayanosii.—Neisseriaceae:—Chromobacterium violaceum;Eikenella corrodens; Kingella denitrificans, Kingella kingae, Kingellaoralis, Kingella potus; Neisseria cinerea; Neisseria elongata; Neisseriaflavescens; Neisseria gonorrhoeae; Neisseria lactamica; Neisseriameningitidis; Neisseria mucosa; Neisseria polysaccharea; Neisseriasicca; Neisseria subflava; Neisseria weaver; Vitreoscillaspp.—Nitrosomonadaceae:—Nitrosomonas eutropha; Nitrosomonas halophila;Nitrosomonas oligotropha.—Pasteurellaceae:—Actinobacillusactinomycetemcomitans; Actinobacillus equuli; Actinobacilluslignieresii; Actinobacillus pleuropneumoniae; Actinobacillus seminis;Actinobacillus succinogenes; Actinobacillus ureae; Aggregatibacteractinomycetemcomitans, Aggregatibacter segnis, Aggregatibacteraphrophilus; Avibacterium avium; Avibacterium endocarditidis;Avibacterium gallinarum; Avibacterium paragallinarum; Avibacteriumvolantium; Bibersteinia trehalose; Gallibacterium anatis; Gallibacteriumgenomospecies 1; Gallibacterium genomospecies 2; Gallibacteriumgenomospecies 3; Gallibacterium group V; Gallibacterium melopsittaci;Gallibacterium salpingitidis; Gallibacterium trehalosifermentans;Haemophilus aegyptius; Haemophilus avium; Haemophilus ducreyi;Haemophilus haemolyticus; Haemophilus influenzae; Haemophilusparahaemolyticus; Haemophilus parainfluenzae; Haemophilus parasuis;Histophilus somni; Mannheimia caviae; Mannheimia glucosida; Mannheimiagranulomatis; Mannheimia haemolytica; Mannheimia ruminalis; Mannheimiavarigena; Nicoletefia semolina; Pasteurella aerogenes; Pasteurellabettyae; Pasteurella caballi; Pasteurella canis; Pasteurella dagmatis;Pasteurella multocida (subspecies multocida, septicum, gallicida);Pasteurella pneumotropica; Pasteurella stomatis; Pasteurellatrehalosi.—Piscirickettsiaceae:—Piscirickettsiasalmonis.—Plesiomonadaceae:—Plesiomonasshigelloides.—Polyangiaceae:—Sorangiumcellulosum.—Porphyromonadaceae:—Dysgonomonas capnocytophagoides;Dysgonomonas gadei; Dysgonomonas hofstadii; Dysgonomonas mossii;Dysgonomonas oryzarvi; Dysgonomonas wimpennyi; Porphyromonasgingivalis.—Prevotellaceae:—Prevotella spp. including Prevotellaintermedia, Prevotella melaninogenica.—Pseudomonadaceae:—Chryseomonasluteola; Pseudomonas aeruginosa; Pseudomonas luteola; Pseudomonasfluorescens; Pseudomonas putida; Pseudomonas stutzeri; Pseudomonasoryzihabitans.—Rhizobiaceae:—Agrobacterium tumefaciens; Rhizobiumradiobacter.—Rickettsiaceae:—Orientia chuto; Orientia tsutsugamushi;Rickettsia aeschlimannii; Rickettsia africae; Rickettsia akari;Rickettsia argasii; Rickettsia asiatica; Rickettsia australis;Rickettsia beffii; Rickettsia canadensis; Rickettsia conorii; Rickettsiacooleyi; Rickettsia felis; Rickettsia heilongjiangensis; Rickettsiahelvetica; Rickettsia honei; Rickettsia hoogstraalii; Rickettsiahulinensis; Rickettsia hulinii; Rickettsia japonica; Rickettsiamarmionii; Rickettsia martinet; Rickettsia massiliae; Rickettsiamonacensis; Rickettsia montanensis; Rickettsia monteiroi; Rickettsiamoreli; Rickettsia parkeri; Rickettsia peacockii; Rickettsia philipii;Rickettsia prowazekii; Rickettsia raoultii; Rickettsia rhipicephali;Rickettsia rickettsii; Rickettsia sibirica subgroup; Rickettsia slovaca;Rickettsia tamurae; Rickettsia typhi.—Shewanellaceae:—Shewanellaputrefaciens.—Sphingomonadaceae:—Sphingobacterium multivorum;Sphingobacterium spiritivorum; Sphingomonaspaucimobilis.—Spirillaceae:—Spirillum minus; Spirillum volutans;Spirillum winogradskyi.—Spirochaetaceae:—Borrelia afzelii; Borreliaanserina; Borrelia bissettii; Borrelia burgdorferi; Borrelia coriaceae;Borrelia duttonii; Borrelia garinii; Borrelia hermsii; Borreliahispanica; Borrelia japonica; Borrelia lonestari; Borrelia lusitaniae;Borrelia miyamotoi; Borrelia parkeri; Borrelia persica; Borreliarecurrentis; Borrelia spielmanii; Borrelia turicatae; Borreliaturicatae; Borrelia valaisiana; Treponema carateum; Treponema pallidumssp. endemicum; Treponema pallidum ssp. pallidum; Treponema pallidumssp. pertenue.—Succinivibrionaceae:—Anaerobiospirillumspp.—Sutterellaceae:—Sutterella spp including Sutterellawadsworthia.—Thermaceae:—Meiothermus spp.—Thermotogaceae:—Thermotoganeapolitana.—Veillonellaceae:—Dialister spp; Megamonas spp; Megasphaeraspp; Pectinatus spp; Pelosinus spp; Propionispora spp; Sporomusa spp;Veillonella spp.; Zymophilus spp.—Vibrionaceae:—Photobacterium damselae;Vibrio adaptatus; Vibrio alginolyticus; Vibrio azasii; Vibriocampbellii; Vibrio cholera; Vibrio damsel; Vibrio fluvialis; Vibriofumisii; Vibrio hoffisae; Vibrio metchnikovii; Vibrio mimicus; Vibrioparahaemolyticus; Vibrio vulnificus.—Wolbachieae:—Wolbachiaspp.—Xanthomonadaceae:—Luteimonas aestuarii; Luteimonas aquatica;Luteimonas composti; Luteimonas lutimaris; Luteimonas marina; Luteimonasmephitis; Luteimonas vadosa; Pseudoxanthomonas broegbernensis;Pseudoxanthomonas japonensis; Stenotrophomonas maltophilia;Stenotrophomonas nitritireducens.

Most preferably, the bacterial agent causing the bacterial infection isgram negative and is selected from the group comprising: Acinetobacterspecies, Aeromonas hydrophila, Citrobacter species, Enterobacterspecies, Escherichia coli, Klebsiella pneumoniae, Morganella morganii,Pseudomonas aeruginosa, and Stenotrophomonas maltophilia.

In another preferred embodiment, the bacteria agent causing thebacterial colonisation or infection is resistant to a conventionalantibiotic used to treat the colonisation or infection. In one preferredembodiment, the bacterial agent is resistant to a compound selected fromthe group comprising: one or more of aminoglycosides (for examplegentamicin, tobramycin, amikacin, or netilmicin); anti-MRSAcephalosporins (for example ceftaroline); antipseudomonalpenicillins+β-lactamase inhibitors (for example ticarcillin-clavulanicacid or piperacillin-tazobactam); carbapenems (for example ertapenem,imipenem, meropenem or doripenem); non-extended spectrum cephalosporins;1st and 2nd generation cephalosporins (for example cefazolin orcefuroxime); extended-spectrum cephalosporins; 3rd and 4th generationcephalosporins (for example cefotaxime or ceftriaxone); cephamycins (forexample cefoxitin or cefotetan); fluoroquinolones (for exampleciprofloxacin); folate pathway inhibitors (for exampletrimethoprim-sulphamethoxazole); glycylcyclines (for exampletigecycline); monobactams (for example aztreonam); penicillins (forexample ampicillin); penicillins+β-lactamase inhibitors (for exampleamoxicillin-clavulanic acid or ampicillin-sulbactam); phenicols (forexample chloramphenicol); phosphonic acids (for example fosfomycin); andpolymyxins (for example colistin); tetracyclines (for exampletetracycline, doxycycline or minocycline). Preferably, the bacterialagent resistant to these compounds is gram negative.

Preferably, the bacterial agent is resistant to a compound selected fromthe group comprising: penicillins, cephalosporins, carbapenems,monobactams and other β-lactam antibiotics, fusidanes, aminoglycosides,fluoroquinolones, streptogramins, tetracyclines, glycylcyclines,chloramphenicol and other phenicols, macrolides and ketolides,lincosamides, oxazolidinones, aminocyclitols, polymyxins, glycopeptides,lipopeptides, bacitracin, mupiricin, pleuromutilins, rifamycins,sulphonamides and trimethoprim. More preferably, the compound isselected from the group comprising: β-lactams, glycopeptides,lipopeptides, macrolides, oxazolidinones and tetracyclines. Preferably,the bacterial agent is resistant to the compound when the compound is ata concentration range selected from the following: 0.001 μg/mL-10,000μg/mL; 0.01 μg/mL-1000 μg/mL; 0.10 μg/mL-100 μg/mL; and 1 μg/mL-50 μg/mL

Most preferably, the bacterial agent causing the bacterial infection isselected from the group comprising, but not limited to, gram positivebacteria. The bacterial agent is most preferably a Gram positivebacterial agent selected from the group comprising Staphylococcusaureus, Staphylococcus pseudintermedius, Streptococcus pneumoniae,Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus uberis,Enterococcus faecium, Enterococcus faecalis, and Clostridium difficile.

In one preferred embodiment, the bacterial agent has no cell wall.Preferably, the bacterial agent is selected from the group comprising:Mycoplasma spp, Mycoplasma agalactiae, Mycoplasma alkalescens,Mycoplasma amphoriforme, Mycoplasma arginini, Mycoplasma bovigenitalum,Mycoplasma bovirhinis, Mycoplasma bovis, Mycoplasma bovoculi, Mycoplasmabuccale, Mycoplasma californicum, Mycoplasma canadense, Mycoplasmacapricolum subsp. capricolum, Mycoplasma capricolum subsp.capripneumoniae, Mycoplasma conjunctivae, Mycoplasma cynos, Mycoplasmadispar, Mycoplasma equigenitalium, Mycoplasma faucium, Mycoplasma felis,Mycoplasma fermentans (incognitus str.), Mycoplasma gallisepticum (MG),Mycoplasma gateae, Mycoplasma genitalium, Mycoplasma haemocanis,Mycoplasma haemofelis, Mycoplasma haemosuis (formerly Eperythrozoonsuis), Mycoplasma hominis, Mycoplasma hyopneumoniae, Mycoplasmahyorhinis, Mycoplasma hyosynoviae, Mycoplasma iowae meleagridis (MM),Mycoplasma iowae, Mycoplasma leachii, Mycoplasma lipophilum, Mycoplasmameleagridis, Mycoplasma mycoides subsp capri, Mycoplasma mycoides subspmycoides, Mycoplasma mycoides subsp. mycoides (such as Contagious bovinepleuropneumonia CBPP), Mycoplasma orale, Mycoplasma ovipneumoniae,Mycoplasma ovis, Mycoplasma penetrans, Mycoplasma pirum, Mycoplasmapneumoniae, Mycoplasma primatum, Mycoplasma putrefaciens, Mycoplasmasalivarium, Mycoplasma spermatophilum, Mycoplasma suis, Mycoplasmasynoviae (MS), Mycoplasma wenyonii, Mycoplasma, Ureaplasma spp,Ureaplasma parvum, Ureaplasma urealyticum, Ureaplasma, and Ureoplasmadiversum.

In another most preferred embodiment, the bacterial agent isStaphylococcus aureus.

In another preferred embodiment, the bacterial agent is resistant to acompound selected from the group comprising: one or more ofaminoglycosides (for example gentamicin); ansamycins (for examplerifampicin); anti-MRSA cephalosporins (for example ceftaroline);anti-staphylococcal β-lactams (or cephamycins) (for example oxacillin orcefoxitin); carbapenems (for example ertapenem, imipenem, meropenem ordoripenem); non-extended spectrum cephalosporins; 1st and 2nd generationcephalosporins (for example cefazolin or cefuroxime); extended-spectrumcephalosporins; 3rd and 4th generation cephalosporins (for examplecefotaxime or ceftriaxone); cephamycins (for example cefoxitin orcefotetan); fluoroquinolones (for example ciprofloxacin ormoxifloxacin); folate pathway inhibitors (for exampletrimethoprim-sulphamethoxazole); fucidanes (for example fusidic acid);glycopeptides (for example vancomycin, teicoplanin or telavancin);glycylcyclines (for example tigecycline); lincosamides (for exampleclindamycin); lipopeptides (for example daptomycin); macrolides (forexample erythromycin); oxazolidinones (for example linezolid ortedizolid); phenicols (for example chloramphenicol); phosphonic acids(for example fosfomycin); streptogramins (for examplequinupristin-dalfopristin); and tetracyclines (for example tetracycline,doxycycline or minocycline). Preferably, the bacterial agent resistantto these compounds is gram positive.

In another most preferred embodiment, the bacterial agent isStreptococcus pneumoniae. The Streptococcus pneumoniae may be a strainthat is resistant to one or more of β-lactams and macrolides.

In another most preferred embodiment, the bacterial agent isStreptococcus pyogenes.

In another most preferred embodiment, the bacterial agent isStreptococcus agalactiae.

In another most preferred embodiment, the bacterial agent is eitherEnterococcus faecium or Enterococcus faecalis. The Enterococcus faeciumor Enterococcus faecalis may be a strain that is resistant to one ormore of aminoglycosides (for example gentamicin (high level) orstreptomycin (for example streptomycin (high level)); carbapenems (forexample imipenem, meropenem or doripenem); fluoroquinolones (for exampleciprofloxacin, levofloxacin or moxifloxacin); glycopeptides (for examplevancomycin or teicoplanin); glycylcyclines (for example tigecycline);lipopeptides (for example daptomycin); oxazolidinones (for examplelinezolid); penicillins (for example ampicillin); streptogramins (forexample quinupristin-dalfopristin); tetracycline (for exampledoxycycline or minocycline).

In another most preferred embodiment, the bacterial agent is Clostridiumdifficile.

The bacterial infection in the subject may cause a disease selected fromthe group comprising, but not limited to, nosocomial pneumonia caused byStaphylococcus aureus (MDR, XDR, PDR or methicillin-susceptible or-resistant strains), or invasive pneumococcal diseases such aspneumonia, bronchitis, acute sinusitis, otitis media, conjunctivitis,meningitis, bacteremia, sepsis, osteomyelitis, septic arthritis,endocarditis, peritonitis, pericarditis, cellulitis, and brain abscesscaused by Streptococcus pneumoniae (including multi-drug resistantstrains [MDRSP] such as those resistant to (3-lactams and macrolides),complicated skin and skin structure infections, including diabetic footinfections, with or without concomitant osteomyelitis, caused byStaphylococcus aureus (methicillin-susceptible and -resistant strains),Streptococcus pyogenes, or Streptococcus agalactiae, uncomplicated skinand skin structure infections caused by Staphylococcus aureus(methicillin-susceptible and -resistant strains) or Streptococcuspyogenes, community-acquired pneumonia caused by Streptococcuspneumoniae (including multi-drug resistant strains [MDRSP], includingcases with concurrent bacteraemia, or Staphylococcus aureus(methicillin-susceptible and -resistant strains) and Staphylococcusaureus bloodstream infections (bacteraemia), including those withright-sided infective endocarditis, caused by methicillin-susceptibleand methicillin-resistant isolates, vancomycin-resistant Enterococcusinfections, including cases with concurrent bacteraemia, and treatmentof Clostridium difficile-associated diarrhea (CDAD).

Gram negative organisms are important causes of many infectious diseasesin humans and other animal species. Bone and joint infections(Gram-negative organisms or mixed bacteria, are an important cause ofvertebral osteomyelitis and septic arthritis), cardiovascular systeminfections (including endocarditis caused by the HACEK group—Haemophilusparainfluenzae, Haemophilus aphrophilus, Aggregatibacteractinomycetemcomitans, Cardiobacterium hominis, Eikenella corrodens,Kingella kingae), central nervous system infections (the commonestcauses of bacterial meningitis are Neisseria meningitidis, Streptococcuspneumoniae and, in nonvaccinated young children, Haemophilus influenzaetype b (Hib), in neonates and infants less than 3 months of age,Streptococcus agalactiae (group B streptococcus), Escherichia coli andother aerobic Gram-negative rods are important pathogens, brain abscessor subdural empyema, the infecting organism(s) vary with the underlyingpredisposing cause but where the likely site of origin is the ear,enteric Gram-negative bacilli are commonly involved), eye infections(common pathogens include Haemophilus influenza, Neisseria gonorrhoeaeor Chlamydia trachomatis), gastrointestinal tract infections (a widerange of pathogens are implicated including enterotoxigenic Escherichiacoli (ETEC), Salmonella, Campylobacter, Shigella, Vibrio cholera andYersinia enterocolitica), genital infections (bacterial vaginosis is apolymicrobial clinical syndrome with high concentrations of anaerobic(eg Mobiluncus species) and other fastidious bacteria (includingGardnerella vaginalis and Atopobium vaginae), and Mycoplasma hominis;non-sexually acquired pelvic inflammatory disease (PID) is usuallycaused by mixed vaginal flora, including anaerobes, facultativeGram-negative bacteria and Mycoplasma hominis, while sexually acquiredPID is usually initiated by C. trachomatis or N. gonorrhoeae withgrowing evidence that M. genitalium infection is involved in asignificant minority of cases), intra-abdominal infections (peritonitisdue to perforated viscus is usually a polymicrobial infection withaerobic and anaerobic bowel flora while spontaneous bacterialperitonitis (SBP) is usually caused by enteric Gram-negative bacilli,such as Escherichia coli and Klebsiella species, Klebsiella pneumoniaeis an increasingly identified cause of liver abscess),community-acquired pneumonia (Mycoplasma pneumoniae, Chlamydophila(Chlamydia) pneumoniae, Chlamydophila (Chlamydia) psittaci, Haemophilusinfluenza, aerobic Gram-negative bacilli including Klebsiella pneumonia,Pseudomonas aeruginosa, Acinetobacter baumannii, Burkholderiapseudomallei), otitis externa (including acute diffuse) (bacterialcultures commonly yield Pseudomonas aeruginosa, Staphylococcus aureus,and Proteus and Klebsiella species), otitis media (including acute)(common bacterial pathogens include Streptococcus pneumoniae,Haemophilus influenzae and Moraxella catarrhalis), sepsis (includingsevere) (including Acinetobacter baumannii, disseminated gonococcalsepsis, Gram-negative enteric bacteria, Neisseria meningitidis(meningococcal sepsis) and Pseudomonas aeruginosa), Systemic infections(Spotted fevers (Rickettsia) and scrub typhus (Orientia), Brucellosis,Cat-scratch disease and other Bartonella infections, Leptospirosis, Lymedisease, Melioidosis, Q fever, Typhoid and paratyphoid fevers (entericfevers), urinary tract infections (acute cystitis, acute pyelonephritis,recurrent urinary tract infections and atheter-associated bacteriuriaand urinary tract infections).

In humans, gram negative bacteria are common causes of intra-abdominalinfections (IAIs), urinary tract infections (UTIs), hospital acquiredpneumonia, and bacteraemia. Escherichia coli (E. coli), Klebsiellapneumoniae (K. pneumoniae), and Pseudomonas aeruginosa (P. aeruginosa)are important pathogens in the hospital setting, accounting for 27% ofall pathogens and 70% of all Gram-negative pathogens causinghealthcare-associated infections [Sievert D M, Ricks P, Edwards J R, etal. Antimicrobial-resistant pathogens associated withhealthcare-associated infections: summary of data reported to theNational Healthcare Safety Network at the Centers for Disease Controland Prevention, 2009-2010. Infect Control Hosp Epidemiol. 2013;34:1-14.].

Gram negative bacteria are showing rising rates of resistance to currenttherapies. The production of extended-spectrum β-lactamase (ESBL)enzymes is a common mechanism of resistance. Rates of ESBL-producing E.coli and K. pneumoniae have risen substantially, with the result thatthese bacteria are increasingly resistant to widely used antimicrobials.

P. aeruginosa is the most common Gram-negative cause of nosocomialpneumonia and the second most common cause of catheter-related UTIs inthe U.S.

E. coli is the most common cause of UTIs. Cases of UTI caused byESBL-producing E. coli and K. pneumonia as well as P. aeruginosa,including MDR strains, are increasing. ESBL-producing E. coli and K.pneumoniae are also frequently isolated in patients with complicated IAI(cIAI).

P. aeruginosa is a clinically challenging and virulent pathogen that canbe a cause of common infections in humans such as nosocomial pneumonia,UTI, IAI, and bloodstream infections. P. aeruginosa is the most commonGram-negative organism causing ventilator associated pneumonia and thesecond most common cause of catheter-associated UTIs.

The increase in the number of infections caused by Gram-negativebacteria is being accompanied by rising rates of resistance. Treatmentoptions to meet this challenge are increasingly limited. There is acritical need for new antibiotics to meet the needs of patients now andin the future.

In another preferred embodiment, robenidine, or a therapeuticallyacceptable salt thereof, is administered together with a compound oragent that removes or substantially removes or reduces the integrity ofthe cell wall of the bacterial agent. As an example, the compound isselected from the group consisting of: β-lactams, fosfomycin, lysozyme,polymyxins and chelating agents such as ethylenediaminetetraacetic acid(EDTA). As an example, the agent is an immunological agent (such as anantibody or vaccine) that reduces the integrity of the cell wall. In onepreferred embodiment, robenidine, or a therapeutically acceptable saltthereof, is administered together with a compound that removes orsubstantially removes or weakens the integrity of the outer cell wall ofa gram negative or positive bacterial agent.

According to another aspect of the invention, there is provided anantibacterial pharmaceutical composition comprising a therapeuticallyeffective amount of robenidine, or a therapeutically acceptable saltthereof.

According to another aspect of the invention, there is provided anantibacterial veterinary composition comprising a therapeuticallyeffective amount of robenidine, or a therapeutically acceptable saltthereof.

The method of treating or preventing a bacterial infection orcolonisation in a subject, may also comprise the administration of thepharmaceutical or veterinary compositions of the invention.

The pharmaceutical composition may optionally include a pharmaceuticallyacceptable excipient or carrier. The veterinary composition mayoptionally include a veterinary acceptable excipient or carrier.

The pharmaceutical or veterinary composition of the invention preferablycontains robenidine, or a pharmaceutically acceptable salt, at aconcentration of selected from the grouped consisting of: 1 mg/g to 500mg/g; 5 mg to 400 mg/g; 10 mg/g to 200 mg/g; 20 mg/g to 100 mg/g; 30mg/g to 70 mg/g; and 40 mg/g to 60 mg/g.

In another embodiment, the pharmaceutical or veterinary compositioncomprises impurities, wherein the quantity of impurities as a percentageof the total weight of the composition is selected from the groupconsisting of: less than 20% impurities (by total weight of thecomposition); less than 15% impurities; less than 10% impurities; lessthan 8% impurities; less than 5% impurities; less than 4% impurities;less than 3% impurities; less than 2% impurities; less than 1%impurities: less than 0.5% impurities; less than 0.1% impurities. In oneembodiment, the pharmaceutical or veterinary composition comprisesmicrobial impurities or secondary metabolites, wherein the quantity ofmicrobial impurities as a percentage of the total weight of thecomposition is selected from the group consisting of: less than 5%; lessthan 4%; less than 3%; less than 2%; less than 1%; less than 0.5%; lessthan 0.1%; less than 0.01%; less than 0.001%. In one embodiment, thepharmaceutical or veterinary composition is sterile and stored in asealed and sterile container. In one embodiment, the pharmaceutical orveterinary composition contains no detectable level of microbialcontamination.

Preferably, the robenidine is pharmaceutical or veterinary grade.Methods to synthesise commercial quantities of robendine are widelyavailable in the art. Commercial quantities of pharmaceutical orveterinary grade robenidine are available from Zhejiang Esigma AnimalHealth Co., Ltd, Haining City, Peoples Republic of China.

The pharmaceutical or veterinary composition of the invention maycomprise a further antimicrobial agent. The further antimicrobial agentmay be an antifungal agent or antibacterial agent. The method oftreating or preventing a bacterial infection or colonisation in asubject, may also comprise the administration of robenidine with afurther antimicrobial agent.

In one embodiment, the antifungal agent is selected from the groupcomprising, but not limited to naturally occurring agents includingEchinocandins (Anidulafungin, Caspofungin, Micafungin), Polyenes(Amphotericin B, Candicidin, Filipin, Fungichromin (Pentamycin),Hachimycin, Hamycin, Lucensomycin, Mepartricin, Natamycin, Nystatin,Pecilocin, Perimycin), and other naturally occurring antifungal agentsincluding Griseofulvin, Oligomycins, Pyrrolnitrin, Siccanin, andViridin. The antifungal agent may be a synthetic compound selected fromthe group comprising, but not limited to Allylamines (Butenafine,Naftifine, Terbinafine) Imidazoles (Bifonazole, Butoconazole,Chlormidazole, Climbazole, Croconazole (Cloconazole), Clotrimazole,Eberconazole, Econazole, Enilconazole, Fenticonazole, Flutrimazole,Fosfluconazole, Isoconazole, Ketoconazole, Lanoconazole, Luliconazole,Miconazole, Neticonazole, Omoconazole, Oxiconazole Nitrate, Parconazole,Sertaconazole, Sulconazole, Tioconazole), Thiocarbamates (Liranaftate,Tolciclate, Tolindate, Tolnaftate), Triazoles (Fluconazole,Isavuconazole, Itraconazole, Posaconazole, Ravuconazole, Saperconazole,Terconazole, Voriconazole), and other synthetic agents such asAcrisorcin, Amorolfine, Bromosalicylchloranilide(Bromochlorosalicylanilide), Buclosamide, Calcium Propionate,Chlorphenesin, Ciclopirox, Cloxyquin (Cloxiquine), Coparaffinate,Exalamide, Flucytosine, Haloprogin, Hexetidine, Loflucarban, Nifuratel,Nifuroxime, Piroctone, Potassium Iodide, Propionic Acid, Pyrithione,Salicylanilide, Sodium Parachlorobenzoate, Sodium Propionate,Sulbentine, Tenonitrozole, Triacetin, Trimetrexate, Undecylenic Acid(Undecenoic Acid), and Zinc Propionate.

The composition of the invention may comprise an antibiotic adjunctselected from the group comprising, but not limited to, β-LactamaseInhibitors (Avibactam, Clavulanic Acid, Sulbactam, Sultamicillin,Tazobactam), Renal Dipeptidase Inhibitors (Cilastatin), and RenalProtectant (Betamipron).

In one embodiment, the composition of the invention comprises a furtherantibiotic selected from the group comprising, but not limited to,2,4-DIAMINOPYRIMIDINES, including Baquiloprim, Brodimoprim, Iclaprim,Ormetoprim, Pyrimethamine, Tetroxoprim, Trimethoprim; AMINOCOUMARINS,including Novobiocin; AMINOCYCLITOLS, including Spectinomycin;AMINOGLYCOSIDES, including Amikacin, Apramycin, Arbekacin, Bekanamycin,Butirosin, Dibekacin, Dihydrostreptomycin, Etimicin, Fortimicins(Astromicin), Framycetin, Gentamicin, Hygromycin B, Isepamicin,Kanamycin, Micronomicin, Neomycin, Netilmicin, Paromomycin, Plazomicin,Ribostamycin, Sisomicin, Streptomycin, Tobramycin, Verdamicin;AMINOMETHYLCYCLINES, including Omadacycline; AMPHENICOLS, includingAzidamfenicol, Chloramphenicol, Florfenicol, Thiamphenicol; ANSAMYCINS,including Rifabutin, Rifamide, Rifampin (Rifampicin), Rifamycin,Rifapentine, Rifaximin; ANTISEPTIC AGENTS, including Acridinederivatives (including acriflavine, aminoacridine, ethacridine,proflavine), Bispyridines (including octenidine dihydrochloride),Brominated salicylanilides (including bromsalans), Chlorhexidine, Phenolderivatives (including thymol and triclosan), Quarternary ammoniumcompounds (including Alkyldimethylethylbenzyl Ammonium Chloride,benzalkonium chloride, cetylpyridinium chloride, benzethonium chloride,cetrimonium); ANTITUBERCULAR AGENTS, including Cycloserine, Delamanid,Ethambutol, Ethionamide, Isoniazid (Ftivazide), Morinamide,p-Aminosalicylic Acid (PAS), Protionamide, Pyrazinamide, Terizidone,Thioacetazone, Tiocarlide; ARSENICALS, including Arsanilic Acid,Roxarsone; BACTERIOCINS, including Nisin, Brilacidin (PMX-30063);β-LACTAM CARBACEPHEMS, including Loracarbef; B-LACTAM CARBAPENEMS,including Biapenem, Doripenem, Ertapenem, Faropenem, Imipenem,Meropenem, Panipenem, Razupenem, Ritipenem, Sulopenem, Tebipenem,Tomopenem; B-LACTAM CEPHALOSPORINS, including Cefacetrile, Cefaclor,Cefadroxil, Cefalexin, Cefaloglycin, Cefalonium, Cefaloridine,Cefalothin, Cefamandole, Cefapirin, Cefatrizine, Cefazaflur, Cefazedone,Cefazolin, Cefcapene, Cefdinir, Cefditoren, Cefepime, Cefetamet,Cefixime, Cefmenoxime, Cefodizime, Cefonicid, Cefoperazone, Ceforanide,Cefoselis, Cefotaxime, Cefotiam, Cefovecin, Cefozopran, Cefpimizole,Cefpiramide, Cefpirome, Cefpodoxime, Cefprozil, Cefquinome, Cefradine,Cefroxadine, Cefsulodin, Ceftaroline, Ceftazidime, Cefteram, Ceftezole,Ceftibuten, Ceftiofur, Ceftizoxime, Ceftobiprole, Ceftolozane,Ceftradine, Ceftrezole, Ceftriaxone, Ceftroxadine, Cefuroxime,Cefuzonam, Pivcefalexin; B-LACTAM CEPHAMYCINS, including Cefbuperazone,Cefmetazole, Cefminox, Cefotetan, Cefoxitin; B-LACTAM MONOBACTAMS,including Aztreonam, Carumonam, Tigemonam; B-LACTAM OXACEPHEMS,including Flomoxef, Latamoxef, Moxalactam; B-LACTAM PENICILLINS,including Amdinocillin (Mecillinam), Amoxicillin, Ampicillin,Apalcillin, Aspoxicillin, Azidocillin, Azlocillin, Bacampicillin,Carbenicillin, Carindacillin, Ciclacillin, Clemizole Penicillin,Clometocillin, Cloxacillin, Cyclacillin, Dicloxacillin, Epicillin,Fenbenicillin, Floxacillin (Flucloxacillin), Hetacillin, Lenampicillin,Mecillinam, Metampicillin, Methicillin Sodium, Mezlocillin, Nafcillin,Oxacillin, Penamecillin, Penethamate Hydriodide, Penicillin G,Penicillin G Benzathine, Penicillin G Procaine, Penicillin N, PenicillinO, Penicillin V, Phenethicillin Potassium, Piperacillin, Pivampicillin,Pivmecillinam, Propicillin, Quinacillin, Sulbenicillin, Sultamicillin,Talampicillin, Temocillin, Ticarcillin; BICYCLOMYCINS, includingBicozamycin; BORON CONTAINING ANTIBACTERIAL AGENTS, including AN3365(aminomethylbenzoxaboroles), GSK2251052 (leucyl-tRNA synthetaseinhibitors); CYCLIC ESTERS, including Fosfomycin; FATTY ACID SYNTHESISINHIBITORS (Fabl), AFN-1252, MUT056399, FAB-001; FLUOROQUINOLONES,including Avarofloxacin, Balofloxacin, Besifloxacin, Chinfloxacin,Cinoxacin, Ciprofloxacin, Clinafloxacin, Danofloxacin, Delafloxacin,Difloxacin, Enoxacin, Enrofloxacin, Finafloxacin, Fleroxacin,Flumequine, Garenoxacin, Gatifloxacin, Gemifloxacin, Grepafloxacin,Ibafloxacin, Levofloxacin, Lomefloxacin, Marbofloxacin, Miloxacin,Moxifloxacin, Nadifloxacin, Norfloxacin, Ofloxacin, Orbifloxacin,Pazufloxacin, Pefloxacin, Pradofloxacin, Prulifloxacin, Rosoxacin,Rufloxacin, Sarafloxacin, Sitafloxacin, Sparfloxacin, Temafloxacin,Tosufloxacin, Trovafloxacin, Zabofloxacin; FUSIDANES, including FusidicAcid; GLYCOLIPODEPSIPEPTIDE, including Ramoplanin; GLYCOPEPTIDES,including Avoparcin, Dalbavancin, Norvancomycin, Oritavancin,Teicoplanin, Telavancin, Vancomycin; GLYCOPHOSPHOLIPIDS, includingBambermycins (bambermycin, moenomycins, flavophospholipol);GLYCYLCYCLINES, including Tigecycline; HYBRIDS, Cadazolid(Oxazolidinone-quinolone), TD-1792 (glycopeptide-cephalosporin);LINCOSAMIDES, including Clindamycin, Lincomycin, Pirlimycin;LIPOPEPTIDES, including Daptomycin, Surotomycin; MACROLIDES, includingAzithromycin, Carbomycin, Cethromycin, Clarithromycin, Dirithromycin,Erythromycin, Fidaxomicin, Flurithromycin, Gamithromycin, Josamycin,Kitasamycin, Leucomycin, Meleumycin, Midecamycins, Miokamycin,Mirosamycin, Oleandomycin, Primycin, Rokitamycin, Rosaramicin,Roxithromycin, Sedecamycin, Solithromycin, Spiramycin, Telithromycin,Terdecamycin, Tildipirosin, Tilmicosin, Troleandomycin, Tulathromycin,Tylosin, Tylvalosin; NITROFURANS, including Furaltadone, Furazidin,Furazolidone, Furazolium Chloride, Nifuratel, Nifurfoline, Nifuroxazide,Nifurpirinol, Nifurtoinol, Nifurzide, Nitrofural, Nitrofurantoin,Nitrofurazone; NITROIMIDAZOLES, including Dimetridazole, Metronidazole,Ornidazole, Ronidazole, Secnidazole, Tinidazole; OLIGOSACCHARIDES,including Avilamycin, Everninomicin; OTHER ANTIBACTERIAL AGENTS,including Auriclosene, Chloroxine, Chlorquinaldol, Clioquinol,Clofoctol, Halquinol, Lotilibcin, Mandelic Acid, Methenamine (hexamine),Nitazole, Nitroxoline, Perchlozone, Taurolidine, Thenoic Acid, Xibornol;OXAZOLIDINONES, including Eperezolid, Linezolid, Posizolid, Radezolid,Sutezolid, Tedizolid (Torezolid); PEPTIDE DEFORMYLASE INHIBITORS,including GSK1322322; PEPTIDES, including Omiganan, Pexiganan;PLEUROMUTILINS, including Retapamulin, Tiamulin, Valnemulin; POLYETHERIONOPHORES, including Laidlomycin, Lasalocid, Maduramicin, Monensin,Narasin, Salinomycin, Semduramicin; POLYMYXINS, including Colistin,Polymyxin B; POLYPEPTIDES, including Amphomycin, Bacitracin,Capreomycin, Enduracidin, Enramycin, Enviomycin, Fusafungine,Gramicidin(s), Iseganan, Magainins, Nosiheptide, Ristocetin,Thiostrepton, Tuberactinomycin, Tyrocidine, Tyrothricin, Viomycin;PSEUDOMONIC ACIDS, including Mupirocin; QUINOLONES, including NalidixicAcid, Nemonoxacin, Oxolinic Acid, Ozenoxacin, Pipemidic Acid, PiromidicAcid; QUINOXALINES, including Carbadox, Olaquindox; RIMINOFENAZINES,including Clofazimine; STATINS, including Atorvastatin, Fluvastatin,Lovastatin, Mevastatin, Pitavastatin, Pravastatin, Rosuvastatin,Simvastatin; STREPTOGRAMINS, including Dalfopristin, Flopristin,Linopristin, Pristinamycin, Quinupristin, Virginiamycin;STREPTOTHRICINS, including Nourseothricin; SULFONAMIDES, includingAcetyl Sulfamethoxypyrazine, Chloramine-B, Chloramine-T, Dichloramine T,Formosulfathiazole, Mafenide, N4-Sulfanilylsulfanilamide,Noprylsulfamide, N-Sulfanilyl-3,4-xylamide, Ormaosulfathiazole,Phthalylsulfacetamide, Phthalylsulfathiazole, Salazosulfadimidine,Succinylsulfathiazole, Sulfabenzamide, Sulfacarbamide, Sulfacetamide,Sulfachlorpyridazine, Sulfachrysoidine, Sulfaclozine, Sulfacytine,Sulfadiazine, Sulfadicramide, Sulfadimethoxine, Sulfadimidine,Sulfadoxine, Sulfaethidole, Sulfaguanidine, Sulfaguanole, Sulfalene,Sulfaloxic Acid, Sulfamerazine, Sulfameter, Sulfamethazine,Sulfamethizole, Sulfamethomidine, Sulfamethoxazole,Sulfamethoxypyridazine, Sulfamethylthiazole, Sulfametopyrazine,Sulfametrole, Sulfamidochrysoidine, Sulfamonomethoxine, Sulfamoxole,Sulfanilamide, Sulfanilylurea, Sulfaperine, Sulfaphenazole,Sulfaproxyline, Sulfapyrazine, Sulfapyridine, Sulfaquinoxaline,Sulfathiazole, Sulfathiourea, Sulfatroxazole, Sulfisomidine,Sulfisoxazole (Sulfafurazole); SULFONES, including Acediasulfone,Dapsone, Glucosulfone Sodium, p-Sulfanilylbenzylamine, Succisulfone,Sulfanilic Acid, Sulfoxone Sodium, Thiazolsulfone; TETRACYCLINES,including Chlortetracycline, Clomocycline, Demeclocycline, Doxycycline,Eravacycline, Guamecycline, Lymecycline, Meclocycline, Methacycline,Minocycline, Oxytetracycline, Penimepicycline, Pipacycline,Rolitetracycline, Sarecycline, Tetracycline.

The composition of the invention may further comprise an excipientselected from the group comprising, but not limited to, binders andcompression aids, coatings and films, colouring agents diluents andvehicles disintegrants, emulsifying and solubilising agents, flavoursand sweeteners, repellents, glidants and lubricants, plasticisers,preservatives, propellants, solvents, stabilisers, suspending agents andviscosity enhancers.

According to a further aspect of the invention, there is provided amedical device when used in a method of treating or preventing abacterial infection in the subject.

According to further aspect of the invention, there is provided amedical device comprising the composition of the invention. Thecomposition of the invention may be any slow release form, and/or in theform of a coating of the medical device.

The medical device may be in a form selected from the group comprising:an implant, a plaster, a bandage, and other dressing applied to abacterial infection in a subject.

According to further aspect of the invention, there is provided a methodof killing bacteria, the method including the step of contacting thebacteria with robenidine, or a therapeutically acceptable salt thereof.

According to further aspect of the invention, there is provided the useof robenidine, or a therapeutically acceptable salt thereof, to killbacteria, said use comprising the step of contacting the bacteria withrobenidine, or a therapeutically acceptable salt thereof.

Terms used herein will have their customary meanings in the art unlessspecified.

BRIEF DESCRIPTION OF THE DRAWINGS

Further features of the present invention are more fully described inthe following description of several non-limiting embodiments thereof.This description is included solely for the purposes of exemplifying thepresent invention. It should not be understood as a restriction on thebroad summary, disclosure or description of the invention as set outabove. The description will be made with reference to the accompanyingdrawings in which:

FIG. 1 shows a table of the Minimum Inhibitory Concentrations for theindividual Staphylococcus aureus isolates according to example 1;

FIG. 2 shows a table of the Minimum Inhibitory Concentrations for theindividual Enterococcus isolates according to example 1;

FIG. 3 shows a table of the Minimum Inhibitory Concentrations for theindividual Streptococcus pneumoniae isolates according to example 1;

FIG. 4 shows a table of the NCL812 MIC₅₀, MIC₉₀, MIC mode and MIC rangefor Australian isolates of MRSA, VRE and Str. pneumoniae according toexample 1. Comparative MIC values for ampicillin are shown inparenthesis;

FIG. 5 shows a table of the Minimum Inhibitory Concentrations values forNCL812 (robenidine) and linezolid against Staphylococcus aureusATCC29213 according to example 2;

FIG. 6 shows a graph of the effect of NCL812 on DNA macromolecularsynthesis in Staphylococcus aureus according to example 2;

FIG. 7 shows a graph of the effect of NCL812 on RNA macromolecularsynthesis in Staphylococcus aureus according to example 2;

FIG. 8 shows a graph of the effect of NCL812 on protein macromolecularsynthesis in Staphylococcus aureus (ATCC29213) according to example 2;

FIG. 9 shows a graph of the effect of NCL812 on cell wall macromolecularsynthesis in Staphylococcus aureus (ATCC29213) according to example 2;

FIG. 10 shows a graph of the effect of NCL812 on lipid macromolecularsynthesis in Staphylococcus aureus (ATCC29213) according to example 2;

FIG. 11 shows a graph summarising the effect of NCL812 on macromolecularsynthesis in Staphylococcus aureus (ATCC29213) according to example 2;

FIG. 12 shows a graph of the effect of NCL812 on ATP release fromStaphylococcus aureus (ATCC29213) according to example 3;

FIG. 13 shows a table of Staphylococcus aureus clone/isolate name, type,source, antibiogram, clindamycin resistance status, multi-locus sequencetype (MLST), staphylococcal cassette chromosome (SCCmec) type, clonalcomplex, Panton-Valentine leukocidin status (PVL), and spa type forisolates used according to example 4. MSSA; methicillin-susceptible S.aureus. HA-MRSA; hospital-acquired methicillin-resistant S. aureus.CA-MRSA; community-associated methicillin-resistant S. aureus. M.B; M.Barton (University of South Australia). G; Gribbles pathology (SouthAustralia). J.P.; J. Perry (University of Adelaide). VIMP; Nares ofstudents from Veterinary Immunology, Microbiology, & Public Health(University of Adelaide). S.P.; S. Polyak (University of Adelaide).G.C.; Geoff Coombs (PathWest Laboratory Medicine, Western Australia).Em; Erythromycin. Ci; Ciprofloxacin. Gn; Gentamicin. Tm; Trimethoprim.Te; Tetracycline. FA; Fusidic Acid. Rf; Rifampicin. Mp; Mupirocin;

FIG. 14 shows a table of the percentage of presumptively identified S.aureus isolates reporting positive to selected phenotypic and genotypictests according to Example 4. HA-MRSA; hospital-acquired S. aureus.CA-MRSA; community-associated S. aureus. S. aureus isolates wereidentified as testing positive to protein A latex agglutination (ProteinA), slide coagulase, Voges-Proskauer and polymyxin B resistance tests,as well as testing positive for polymerase chain reaction (PCR) andreal-time PCR amplification of the spa gene. Methicillin-resistant S.aureus isolates were identified as isolates testing positive to thecriteria described above, as well as positive for PCR and real-time PCRof the mecA gene;

FIG. 15 shows a table of the resistance of S. aureus isolates toantibacterial agents using the Kirby-Bauer disc diffusion methodaccording to Example 4. HA-MRSA; hospital-acquired methicillin-resistantS. aureus. CA-MRSA; community-associated methicillin-resistant S.aureus;

FIG. 16 shows a table of the number and percentage of identified mecgene complexes in 20 S. aureus strains classified asmethicillin-resistant according to Example 4. Respective staphylococcalcassette chromosome (SCCmec) complexes and types expressing phenotypicresistance to oxacillin and cefotetan are indicated as well as real-timemecA status, and the average negative dF/dT peak obtained from meltingpoint analysis from real-time PCR of the mecA gene. Figures inparentheses indicate percentages;

FIG. 17 shows a graph showing the average melting point peaks for thenegative derivative plot −dF/dT after real-time polymerase chainreaction of the mecA gene in methicillin-resistant S. aureus isolatesgrouped by mec gene complexes, A (n=4), B (n=10), C2 (n=4) andunclassified (n=2). Groups indicated with different superscripts aresignificantly different (P<0.05), according to Example 4;

FIG. 18 shows a table of the characteristics of antibacterial NCL812 andthe β-lactam antibacterial ampicillin according to Example 4, detailingantibacterial solubility in dimethyl sulfoxide (DMSO), solubility incation-adjusted Mueller-Hinton II broth (CAMHB), and average minimuminhibitory concentrations (MIC) (μg/ml at 24-h) againstmethicillin-resistant S. aureus (MRSA) determined from preliminarystudies and those determined during this present study. ATCC 49775;methicillin-susceptible S. aureus isolate and ATCC control strain.MRSA580; methicillin-resistant S. aureus isolate #580. MRSA698;methicillin-resistant S. aureus isolate #698;

FIG. 19 shows a table of in vitro activities of the novel antibacterialNCL812 and the β-lactam antibacterial ampicillin against S. aureusclinical isolates according to example 4. HA-MRSA; hospital-acquiredmethicillin-resistant S. aureus. CA-MRSA; community-associatedmethicillin-resistant S. aureus. MIC; minimum inhibitory concentration(μg/ml). MBC; minimum bactericidal concentration (μg/ml). MIC/MBCrange;minimum and maximum MIC/MBC for all isolates. MIC/MBC50; MIC/MBC atwhich 50% of isolates are inhibited. MIC/MBC90; MIC/MBC at which 90% ofisolates are inhibited;

FIG. 20 shows a graph of the optical densities of the unsupplementedgrowth control, ampicillin and different concentrations of antibacterialagent NCL812 against methicillin-susceptible S. aureus ATCC 49775 usingbroth microdilution methodology according to example 4. Theconcentrations of NCL812 tested were at the MIC and four times the MICdetermined under test conditions, up to 24-h incubation. Ampicillin wastested at the MIC. Bactericidal activity was tested at 0-, 1-, 2-, 4-,8-, 12-, and 24-h for antibacterials;

FIG. 21 shows a graph of kill kinetic curves for methicillin-susceptibleS. aureus ATCC 49775 demonstrating bactericidal activity of NCL812 usingthe Clinical and Laboratory Standards Institute macrodilutionmethodology in a 10-ml vial according to example 4. The concentrationsof antibacterials tested were at 1× and 4× the MIC determined under testconditions. Bactericidal activity was determined at 0-, 1-, 2-, 4-, 8-,12- and 24-h after antibacterial addition. Bactericidal activity wasdefined as a 3-log 10 (99.9%) decrease in the number viable bacteriafrom the initial inoculum size;

FIG. 22 shows a table of the antibacterial susceptibility of 20 S.pneumoniae isolates for six different antibacterials according toexample 5;

FIG. 23 shows a graph indicating the change of pH during macro-brothdilution assay for S. pneumoniae strain D39 exposed to 4 μg/ml in NCL812and 0.0023 μg/ml ampicillin according to example 5;

FIG. 24 shows a graph illustrating the 48-hour time-kill of S.pneumoniae strain D39 treated with NCL812 according to example 5;

FIG. 25 shows a graph illustrating in the 14-hour time-kill of S.pneumoniae strain D39 treated with NCL812 according to example 5;

FIG. 26 shows a graph illustrating the 14-hour time-kill of S.pneumoniae strain D39 treated with ampicillin according to example 5;

FIG. 27 shows a graph illustrating the 12-hour time-kill of S.pneumoniae strain D39 treated with NCL812, adopted from FIG. 40according to example 5;

FIG. 28 shows a graph illustrating the 48-hour time-kill of S.pneumoniae strain D39 treated with ampicillin according to example 5;

FIG. 29 shows a graph illustrating the 48-hour time-kill of S.pneumoniae strain D39 treated with erythromycin according to example 5;

FIG. 30 shows a graph illustrating the 48-hour time-kill of S.pneumoniae strain D39 treated with NCL812 and 5% choline chlorideaccording to example 5;

FIG. 31 shows a graph illustrating the 12-hour time-kill of S.pneumoniae strain D39 treated with NCL812 and 5% choline chlorideaccording to example 5;

FIG. 32 shows a graph illustrating the relative minimum bactericidalconcentration (MBC) of S. pneumoniae strain D39 treated with ampicillinover a 48-hour time period according to example 5;

FIG. 33 shows a graph illustrating the relative MBC for S. pneumoniaestrain D39 treated with erythromycin over a 48-hour time periodaccording to example 5;

FIG. 34 shows a graph illustrating the viable count (log₁₀ CFU/ml) of S.pneumoniae strain D39 treated with NCL812 from a macro-broth dilution oftime-kill over 24 hours according to example 5;

FIG. 35 shows a graph illustrating the viable count (log₁₀ CFU/ml) of S.pneumoniae strain D39 treated with ampicillin from a macro-brothdilution of time-kill over 24 hours according to example 5;

FIG. 36 is a bar graph illustrating the mean cell membrane thickness oftreated and untreated D39 according to example 5;

FIG. 37 is a bar graph illustrating the mean width of periplasmic spaceof treated (16 μg/ml NCL812) and untreated D39 samples according toexample 5;

FIG. 38 is a table showing the Staphylococcus pseudintermedius isolatestested according to example 6;

FIG. 39 is a table showing the antibiotic resistance profile of theStaphylococcus pseudintermedius isolates tested according to example 6.

FIG. 40 is a graph illustrating the effectiveness of NCL812 againstgram-negative E. coli spheroplasts according to example 7;

FIG. 41 is a graph illustrating the cumulative release of NCL812 fromFormulation B according to example 9;

FIG. 42 shows the kill kinetics assay of Staphylococcus aureus KC01 atdifferent concentrations of NCL812, up to 24 h incubation according toexample 11; and

FIG. 43 shows the kill kinetics assay of Enterococcus faecalis USA01 atdifferent concentrations of NCL812, up to 24 h incubation according toexample 11.

DESCRIPTION OF EMBODIMENTS General

Before describing the present invention in detail, it is to beunderstood that the invention is not limited to particular exemplifiedmethods or compositions disclosed herein. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments of the invention only, and is not intended to belimiting.

All publications referred to herein, including patents or patentapplications, are incorporated by reference in their entirety. However,applications that are mentioned herein are referred to simply for thepurpose of describing and disclosing the procedures, protocols, andreagents referred to in the publication which may have been used inconnection with the invention. The citation of any publications referredto herein is not to be construed as an admission that the invention isnot entitled to antedate such disclosure by virtue of prior invention.

In addition, the carrying out of the present invention makes use of,unless otherwise indicated, conventional microbiological techniqueswithin the skill of the art. Such conventional techniques are known tothe skilled worker.

As used herein, and in the appended claims, the singular forms “a”,“an”, and “the” include the plural unless the context clearly indicatesotherwise.

Unless otherwise indicated, all technical and scientific terms usedherein have the same meanings as commonly understood by one of ordinaryskill in the art to which this invention belongs. Although any materialsand methods similar to, or equivalent to, those described herein may beused to carry out the present invention, the preferred materials andmethods are herein described.

The invention described herein may include one or more ranges of values(e.g. size, concentration, dose etc). A range of values will beunderstood to include all values within the range, including the valuesdefining the range, and values adjacent to the range that lead to thesame or substantially the same outcome as the values immediatelyadjacent to that value which define the boundary of the range.

The pharmaceutical for veterinary compositions of the invention may beadministered in a variety of unit dosages depending on the method ofadministration, target site, physiological state of the patient, andother medicaments administered. For example, unit dosage form suitablefor oral administration include solid dosage forms such as powder,tablets, pills, and capsules, and liquid dosage forms, such as elixirs,syrups, solutions and suspensions. The active ingredients may also beadministered parenterally in sterile liquid dosage forms. Gelatincapsules may contain the active ingredient and inactive ingredients suchas powder carriers, glucose, lactose, sucrose, mannitol, starch,cellulose or cellulose derivatives, magnesium stearate, stearic acid,sodium saccharin, talcum, magnesium carbonate, and the like.

The phrase “therapeutically effective amount” as used herein refers toan amount sufficient to inhibit bacterial growth associated with abacterial infection or colonisation. That is, reference to theadministration of the therapeutically effective amount of robenidineaccording to the methods or compositions of the invention refers to atherapeutic effect in which substantial bacteriocidal or bacteriostaticactivity causes a substantial inhibition of bacterial infection. Theterm “therapeutically effective amount” as used herein, refers to asufficient amount of the composition to provide the desired biological,therapeutic, and/or prophylactic result. The desired results includeelimination of bacterial infection or colonisation or reduction and/oralleviation of the signs, symptoms, or causes of a disease, or any otherdesired alteration of a biological system. An effective amount in anyindividual case may be determined by one of ordinary skill in the artusing routine experimentation. In relation to a pharmaceutical orveterinary composition, effective amounts can be dosages that arerecommended in the modulation of a diseased state or signs or symptomsthereof. Effective amounts differ depending on the composition used andthe route of administration employed. Effective amounts are routinelyoptimized taking into consideration pharmacokinetic and pharmacodynamiccharacteristics as well as various factors of a particular patient, suchas age, weight, gender, etc and the area affected by disease or diseasecausing microbes.

As referred to herein, the terms “treatment” or “treating” refers to thefull or partial removal of the symptoms and signs of the condition. Forexample, in the treatment of a bacterial infection or colonisation, thetreatment completely or partially removes the signs of the infection.Preferably in the treatment of infection, the treatment reduces oreliminates the infecting bacterial pathogen leading to microbial cure.

As referred to herein, the term “bacteria” refers to members of a largedomain of prokaryotic microorganisms. Typically a few micrometres inlength, bacteria have a number of shapes, ranging from spheres to rodsand spirals and can be present as individual cells or present in linearchains or clusters of variable numbers and shape. Preferably the terms“bacteria” and its adjective “bacterial” refer to bacteria such as theGram positive Staphylococcus spp, Streptococcus spp, Bacillus spp,Enterococcus spp, Listeria spp, Mycoplasma spp, and anaerobic bacteria;Gram negative Escherichia coli, Enterobacter spp, Klebsiella spp andPseudomonas spp; and the cell wall free bacteria such as Mycoplasma sppand Ureaplasma spp. The terms may refer to an antibiotic-sensitivestrain or an antibiotic-resistant strain. In a preferred embodiment, theterms refer to MRSA or MRSP. In another preferred embodiment, the termsrefer to MDR Staphylococcus spp, Streptococcus spp, Enterococcus spp,Clostridium difficile, Escherichia coli, Enterobacter spp, Klebsiellaspp and Pseudomonas spp.

Referred to herein, the term “methicillin-resistant bacteria” (such asmethicillin-resistant Staphylococcus) refers a bacteria isolate thatdemonstrates resistance at any dose to all 6 lactams includingpenicillins, carbapenems and first to fourth generation cephalosporins,but not to the fifth generation anti-MRSA cephalosporins (for exampleceftaroline). Multidrug-resistant (MDR) is defined as acquirednon-susceptibility to at least one agent in three or more antimicrobialcategories, extensively drug-resistant (XDR) is defined asnon-susceptibility to at least one agent in all but two or fewerantimicrobial categories (i.e. bacterial isolates remain susceptible toonly one or two categories) and pandrug-resistant (PDR) is defined asnon-susceptibility to all agents in all antimicrobial categoriescurrently available.

An example of susceptible, MDR, XDR and PDR bacteria includes thefollowing. Wild type, antibacterial unexposed isolates of Staphylococcusaureus that are likely to be susceptible to all of the followingantibacterial categories (and agents): aminoglycosides (for examplegentamicin); ansamycins (for example rifampicin); anti-MRSAcephalosporins (for example ceftaroline); anti-staphylococcal β-lactams(or cephamycins) (for example oxacillin or cefoxitin); carbapenems (forexample ertapenem, imipenem, meropenem or doripenem); non-extendedspectrum cephalosporins; 1st and 2nd generation cephalosporins (forexample cefazolin or cefuroxime); extended-spectrum cephalosporins; 3rdand 4th generation cephalosporins (for example cefotaxime orceftriaxone); cephamycins (for example cefoxitin or cefotetan);fluoroquinolones (for example ciprofloxacin or moxifloxacin); folatepathway inhibitors (for example trimethoprim-sulphamethoxazole);fucidanes (for example fusidic acid); glycopeptides (for examplevancomycin, teicoplanin or telavancin); glycylcyclines (for exampletigecycline); lincosamides (for example clindamycin); lipopeptides (forexample daptomycin); macrolides (for example erythromycin);oxazolidinones (for example linezolid or tedizolid); phenicols (forexample chloramphenicol); phosphonic acids (for example fosfomycin);streptogramins (for example quinupristin-dalfopristin; and tetracyclines(for example tetracycline, doxycycline or minocycline). Isolates thatare non-susceptible to more than one agent in more than threeantimicrobial categories are classified as MDR (all MRSA, for example,meet the definition of MDR). Isolates that are non-susceptible to morethan one agent in all but one or two antimicrobial categories areclassified as XDR. Isolates that are non-susceptible to all listedantibacterial agents are PDR.

Pharmaceutically and veterinary acceptable salts include salts whichretain the biological effectiveness and properties of the compounds ofthe present disclosure and which are not biologically or otherwiseundesirable. In many cases, the compounds disclosed herein are capableof forming acid and/or base salts by virtue of the presence of aminoand/or carboxyl groups or groups similar thereto. Acceptable baseaddition salts can be prepared from inorganic and organic bases. Saltsderived from inorganic bases, include by way of example only, sodium,potassium, lithium, ammonium, calcium and magnesium salts. Salts derivedfrom organic bases include, but are not limited to, salts of primary,secondary and tertiary amines, such as by way of example only, alkylamines, dialkyl amines, trialkyl amines, substituted alkyl amines,di(subsrituted alkyl) amines, tri(substituted alkyl) amines, alkenylamines, dialkenyl amines, trialkenyl amines, substituted alkenyl amines,di(substituted alkenyl) amines, tri(substituted alkenyl) amines,cycloalkyl amines, di(cycloalkyl) amines, tri(cycloalkyl) amines,substituted cycloalkyl amines, disubstituted cycloalkyl amines,trisubstituted cycloalkyl amines, cycloalkenyl amines, di(cycloalkenyl)amines, tri(cycloalkenyl) amines, substituted cycloalkenyl amines,disubstituted cycloalkenyl amines, trisubstituted cycloalkenyl amines,aryl amines, diaryl amines, triaryl amines, heteroaryl amines,diheteroaryl amines, triheteroaryl amines, heterocyclic amines,diheterocyclic amines, triheterocyclic amines, mixed di- and tri-amineswhere at least two of the substituents on the amine are different andare selected from the group consisting of alkyl, substituted alkyl,alkenyl, substituted alkenyl, cycloalkyl, substituted cycloalkyl,cycloalkenyl, substituted cycloalkenyl, aryl, heteroaryl, heterocyclic,and the like. Also included are amines where the two or threesubstituents, together with the amino nitrogen, form a heterocyclic orheteroaryl group.

Pharmaceutically and veterinary acceptable acid addition salts may beprepared from inorganic and organic acids. The inorganic acids that canbe used include, by way of example only, hydrochloric acid, hydrobromicacid, sulfuric acid, nitric acid, phosphoric acid, and the like. Theorganic acids that can be used include, by way of example only, aceticacid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, malicacid, malonic acid, succinic acid, maleic acid, fumaric acid, tartaricacid, citric acid, benzoic acid, cinnamic acid, mandelic acid,methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid,salicylic acid, and the like.

The pharmaceutically or veterinary acceptable salts of the compoundsuseful in the present disclosure can be synthesized from the parentcompound, which contains a basic or acidic moiety, by conventionalchemical methods. Generally, such salts can be prepared by reacting thefree acid or base forms of these compounds with a stoichiometric amountof the appropriate base or acid in water or in an organic solvent, or ina mixture of the two; generally, nonaqueous media like ether, ethylacetate, ethanol, isopropanol, or acetonitrile are preferred. Lists ofsuitable salts are found in Remington's Pharmaceutical Sciences. 17thed., Mack Publishing Company, Easton, Pa. (1985), p. 1418, thedisclosure of which is hereby incorporated by reference. Examples ofsuch acceptable salts are the iodide, acetate, phenyl acetate,trifluoroacetate, acryl ate, ascorbate, benzoate, chlorobenzoate,dinitrobenzoate, hydroxybenzoate, methoxybεnzoate, methylbenzoate,o-acetoxybenzoate, naphthalene-2-benzoate, bromide, isobutyrate,phenylbutyrate, γ-hydroxybutyrate, β-hydroxybutyrate, butyne-1,4-dioate,hexyne-1,4-dioate, hexyne-1,6-dioate, caproate, caprylate, chloride,cinnamate, citrate, decanoate, formate, fumarate, glycollate,heptanoate, hippurate, lactate, malate, maleate, hydroxymaleate,malonate, mandelate, mesylate, nicotinate, isonicotinate, nitrate,oxalate, phthalate, terephthalate, phosphate, monohydrogenphosphate,dihydrogenphosphate, metaphosphate, pyrophosphate, propiolate,propionate, phenylpropionate, salicylate, sebacate, succinate, suberate,sulfate, bisulfate, pyrosulfate, sulfite, bisulfite, sulfonate,benzenesulfonate, p-bromophenylsulfonate, chlorobenzenesulfonate,propanesulfonate, ethanesulfonate, 2-hydroxyethanesulfonate,merhanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate,p-toluenesulfonate, xylenesulfonate, tartarate, and the like.

The pharmaceutical or veterinary compositions of the invention may beformulated in conventional manner, together with other pharmaceuticallyacceptable excipients if desired, into forms suitable for oral,parenteral, or topical administration. The modes of administration mayinclude parenteral, for example, intramuscular, subcutaneous andintravenous administration, oral administration, topical administrationand direct administration to sites of infection such as intraocular,intraaural, intrauterine, intranasal, intramammary, intraperitoneal andintralesional.

The pharmaceutical or veterinary compositions of the invention may beformulated for oral administration. Traditional inactive ingredients maybe added to provide desirable colour, taste, stability, bufferingcapacity, dispersion, or other known desirable features. Examplesinclude red iron oxide, silica gel, sodium laurel sulphate, titaniumdioxide, edible white ink, and the like. Conventional diluents may beused to make compressed tablets. Both tablets and capsules may bemanufactured as sustained-release compositions for the continual releaseof medication over a period of time. Compressed tablets may be in theform of sugar coated or film coated tablets, or enteric-coated tabletsfor selective disintegration in the gastrointestinal tract. Liquiddosage forms for oral administration may contain colouring and/orflavouring to increase patient compliance. As an example, the oralformulation comprising NCL812 may be a tablet comprising anyone, or acombination of, the following excipients: calcium hydrogen phosphatedehydrate, microcrystalline cellulose, lactose, hydroxypropyl methylcellulose, and talc.

The compositions described herein may be in the form of a liquidformulation. Examples of preferred liquid compositions includesolutions, emulsions, injection solutions, solutions contained incapsules. The liquid formulation may comprise a solution that includes atherapeutic agent dissolved in a solvent. Generally, any solvent thathas the desired effect may be used in which the therapeutic agentdissolves and which can be administered to a subject. Generally, anyconcentration of therapeutic agent that has the desired effect can beused. The formulation in some variations is a solution which isunsaturated, a saturated or a supersaturated solution. The solvent maybe a pure solvent or may be a mixture of liquid solvent components. Insome variations the solution formed is an in situ gelling formulation.Solvents and types of solutions that may be used are well known to thoseversed in such drug delivery technologies.

The composition described herein may be in the form of a liquidsuspension. The liquid suspensions may be prepared according to standardprocedures known in the art. Examples of liquid suspensions includemicro-emulsions, the formation of complexing compounds, and stabilisingsuspensions. The liquid suspension may be in undiluted or concentratedform. Liquid suspensions for oral use may contain suitablepreservatives, antioxidants, and other excipients known in the artfunctioning as one or more of dispersion agents, suspending agents,thickening agents, emulsifying agents, wetting agents, solubilisingagents, stabilising agents, flavouring and sweetening agents, colouringagents, and the like. The liquid suspension may contain glycerol andwater.

The composition described herein may be in the form of an oral paste.The oral paste may be prepared according to standard procedures known inthe art.

The composition is described herein may be in the form of a liquidformulation for injection, such as intra-muscular injection, andprepared using methods known in the art. For example, the liquidformulation may contain polyvinylpyrrolidone K30 and water.

The composition is described herein may be in the form of topicalpreparations. The topical preparation may be in the form of a lotion ora cream, prepared using methods known in the art. For example, a lotionmay be formulated with an aqueous or oily base and may include one ormore excipients known in the art, functioning as viscosity enhancers,emulsifying agents, fragrances or perfumes, preservative agents,chelating agents, pH modifiers, antioxidants, and the like. For example,the topical formulation comprising NCL812 may be a gel comprisinganyone, or a combination of, the following excipients: PEG 4000, PEG200, glycerol, propylene glycol. The NCL812 compound may further beformulated into a solid dispersion using SoluPlus (BASF,www.soluplys.com) and formulated with anyone, or a combination of, thefollowing excipients: PEG 4000, PEG 200, glycerol, propylene glycol.

For aerosol administration, the composition is of the invention they beprovided in finely divided form together with a non-toxic surfactant anda propellant. The surfactant is preferably soluble in the propellant.Such surfactants may include esters or partial esters of fatty acids.

The compositions of the invention may alternatively be formulated usingnanotechnology drug delivery techniques such as those known in the art.Nanotechnology-based drug delivery systems have the advantage ofimproving bioavailability, patient compliance and reducing side effects.

The formulation of the composition of the invention includes thepreparation of nanoparticles in the form of nanosuspensions ornanoemulsions, based on compound solubility. Nanosuspensions aredispersions of nanosized drug particles prepared by bottom-up ortop-down technology and stabilised with suitable excipients. Thisapproach may be applied to robenidene which has poor aqueous and lipidsolubility in order to enhance saturation solubility and improvedissolution characteristics. An example of this technique is set out inSharma and Garg (2010) (Pure drug and polymer-based nanotechnologies forthe improved solubility, stability, bioavailability, and targeting ofanti-HIV drugs. Advanced Drug Delivery Reviews, 62: p. 491-502).Saturation solubility will be understood to be a compound-specificconstant that depends on temperature, properties of the dissolutionmedium, and particle size (<1-2 μm).

The composition of the invention may be provided in the form of anansuspension. For nanosuspensions, the increase in the surface area maylead to an increase in saturation solubility. Nanosuspensions arecolloidal drug delivery systems, consisting of particles below 1 μm.Compositions of the invention may be in the form of nanosuspensionsincluding nanocrystalline suspensions, solid lipid nanoparticles (SLNs),polymeric nanoparticles, nanocapsules, polymeric micelles anddendrimers. Nanosuspensions may be prepared using a top-down approachwhere larger particles may be reduced to nanometre dimensions by avariety of techniques known in the art including wet-milling andhigh-pressure homogenisation. Alternatively, nanosuspensions may beprepared using a bottom-up technique where controlled precipitation ofparticles may be carried out from solution.

The composition of the invention may be provided in the form of ananoemulsion. Nanoemulsions are typically clear oil-in-water orwater-in-oil biphasic systems, with a droplet size in the range of100-500 nm, and with compounds of interest present in the hydrophobicphase. The preparation of nanoemulsions may improve the solubility ofrobenidine described herein, leading to better bioavailability.Nanosized suspensions may include agents for electrostatic or stericstabilisation such as polymers and surfactants. Compositions in the formof SLNs may comprise biodegradable lipids such as triglycerides,steroids, waxes and emulsifiers such as soybean lecithin, egg lecithin,and poloxamers. The preparation of a SLN preparation may involvedissolving/dispersing drug in melted lipid followed by hot or coldhomogenisation. If hot homogenisation is used, the melted lipidic phasemay be dispersed in an aqueous phase and an emulsion prepared. This maybe solidified by cooling to achieve SLNs. If cold homogenisation isused, the lipidic phase may be solidified in liquid nitrogen and groundto micron size. The resulting powder may be subjected to high-pressurehomogenisation in an aqueous surfactant solution.

Robenidine as described herein may be dissolved in oils/liquid lipidsand stabilised into an emulsion formulation. Nanoemulsions may beprepared using high- and low-energy droplet reduction techniques.High-energy methods may include high-pressure homogenisation,ultrasonication and microfluidisation. If the low-energy method is used,solvent diffusion and phase inversion will generate a spontaneousnanoemulsion. Lipids used in nanoemulsions may be selected from thegroup comprising triglycerides, soybean oil, safflower oil, and sesameoil. Other components such as emulsifiers, antioxidants, pH modifiersand preservatives may also be added.

The composition may be in the form of a controlled-release formulationmay include a degradable or non-degradable polymer, hydrogel, organogel,or other physical construct that modifies the release of the polyetherionophore. It is understood that such formulations may includeadditional inactive ingredients that are added to provide desirablecolour, stability, buffering capacity, dispersion, or other knowndesirable features. Such formulations may further include liposomes,such as emulsions, foams, micelles, insoluble monolayers, liquidcrystals, phospholipid dispersions, lamellar layers and the like.Liposomes for use in the invention may be formed from standardvesicle-forming lipids, generally including neutral and negativelycharged phospholipids and a sterol, such as cholesterol.

The formulations of the invention may have the advantage of increasedsolubility and/or stability of NCL812, particularly for thoseformulations prepared using nanotechnology techniques. Such increasedstability and/or stability of NCL812 may improve bioavailability andenhance drug exposure for oral and/or parenteral dosage forms.

Throughout this specification, unless the context requires otherwise,the word “comprise” or variations such as “comprises” or “comprising”,will be understood to imply the inclusion of a stated integer or groupof integers but not the exclusion of any other integer or group ofintegers.

EXAMPLES Example 1

The minimum inhibitory concentrations (MIC) for NCL812 inmethicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistantEnterococcus spp. (VRE) and Streptococcus pneumoniae.

In this example and other examples in the specification, the term NCL812is used to indicate robenidine.

This study was undertaken to determine minimum inhibitory concentrations(MIC) for a new antibacterial agent, NCL812. The antibacterial agentrepresents a potentially new class of drug with a perceived narrowspectrum of activity against bacteria and a novel mechanism of action.This study focused on recent isolates of three major opportunisticpathogens of humans where the development of antibacterial resistance toexisting antibacterial classes is problematic: methicillin-resistantStaphylococcus aureus (MRSA), vancomycin-resistant Enterococcus spp.(VRE) and Streptococcus pneumoniae.

Materials and Methods Bacterial Isolate Collection and Identification

Sixty one test isolates were sourced from clinical diagnosticmicrobiology laboratories. The MRSA isolates were originally cultured onselective Brilliance MRSA Chromogenic Agar (Oxoid). Suspect colonieswere selected on the basis of their colony appearance on this agar andidentification as Staphylococcus aureus was determined using colonycharacteristics on non-selective Sheep Blood Agar and phenotypiccharacteristics such as Gram stain, positive catalase test, positivecoagulase test (tube coagulase test using rabbit plasma) and clumpingfactor (agglutination with the Oxoid Staphytect latex test), positiveVoges Proskauer test, and the ability to produce acid from trehalose. Apositive cefoxitin resistance screen confirmed the isolates as MRSA. AllEnterococcus isolates underwent a standard biochemical identification.Biochemical profiling provisionally identified four of the VRE isolatesas Enterococcus faecalis and the remainder as Enterococcus faecium,however this is not 100% reliable for human Enterococcus strains andfull biochemical profiling using API-ZYM profiling will be undertaken to100% confirm identity. All Str. pneumoniae isolates were identified onthe basis of standard biochemical profiling.

Preparation of Antimicrobials

Analytical grade NCL812 (batch 20081214) with a defined potency of 1000mg/g (ie 100%) was obtained The powder was stored at a temperature of−20° C. at the study site, in a locked freezer. Aliquots (1 ml) of stocksolution (25.6 mg/ml) were prepared in DMSO and stored at −80° C. anddefrosted immediately before use.

Minimum Inhibitory Concentration Assay

Minimum inhibitory concentration tests were performed according to CLSIStandards (CLSI 2008). 90 μL of one of the test compound solutions, orampicillin, was added to the end column of a 96 well plate thatcontained 90 μL of CAMHB in each well. The solutions were then seriallydiluted across the row, leaving 2 columns for positive and negativecontrols. A bacterial suspension was prepared by adding fresh coloniesobtained from an overnight culture on Sheep Blood Agar (SBA) to a 9.1g/L saline solution. This suspension was adjusted to a concentration ofbetween 4×10⁸ and 5×10⁸ CFU/mL. Concentration of the suspension wasdetermined by measuring optical density (OD) using a spectrophotometerat a wavelength of 600 nm where the correct concentration was determinedto have an optical density of between 1.00 and 1.20. One millilitre ofthis suspension was added to 9 mL of saline before being added to allwells, excluding the negative control wells, in 10 μL volumes giving afinal concentration of between 4×10⁵ and 5×10⁵ CFU/mL in each well. Thetests were then incubated for 24 hours at 37° C. and then assessed bothvisually and using OD readings from a microplate reader at a wavelengthof 600 nm. These tests were performed in duplicate but repeated ifdiscrepancies were observed.

The minimum inhibitory concentration (MIC) was determined to be thelowest concentration of antibiotic that prevented growth of bacteriaboth visually and using OD readings. Direct statistical comparisonsbetween the test compounds and ampicillin could not be performed inlight of confidential information restrictions, such as restrictions ondisclosure of information relating to the compound structure, such asmolecular weight. Instead, MIC values were collated and used todetermine the lowest concentration of each compound that was effectiveagainst 50% and 90% of isolates, referred to as the MIC₅₀ and MIC₉₀respectively. These values, as well as the range of MIC values, werethen used for direct comparisons between test compounds and for generalcomparisons with ampicillin.

Results

Ampicillin MIC values obtained for the ATCC control strains were withinthe normal range expected on the basis of CLSI recommendations. TheNCL812 and ampicillin MIC values for each isolate are indicated in FIG.1 (MRSA isolates), FIG. 2 (VRE isolates) and FIG. 3 (Str. pneumoniaeisolates).

The pooled MIC50, MIC90, MIC mode and MIC range for NCL812 for each ofthe species of bacteria tested are shown in FIG. 4.

NCL812 MIC values were remarkably consistent within and between each ofthe three species. MIC50 and MIC90 values were both equal (4 μg/ml) forMRSA, VRE and Str. pneumoniae isolates, with less than 10% of isolatesshowing MIC values either 1-2 dilutions below or only one dilution abovethis figure.

Example 2

Effect of NCL812 on Staphylococcus aureus Macromolecular Synthesis

Materials and Methods Test Compounds

Test compound NCL812 was transported to the experimental facility underconditions of ambient temperature and then stored at 2-8° C. untilassayed. Stock solutions were made by dissolving NCL812 dry powder in100% DMSO to a concentration of 6,400 μg/ml.

Minimal Inhibitory Concentration Testing

The MIC assay method followed the procedure described by the Clinicaland Laboratory Standards Institute, or CLSI (Clinical and LaboratoryStandards Institute). Methods for Dilution Antimicrobial SusceptibilityTests for Bacteria That Grow Aerobically; Approved Standard—EighthEdition. CLSI document M07-A8 [ISBN 1-56238-689-1]. Clinical andLaboratory Standards Institute, 940 West Valley Road, Suite 1400, Wayne,Pa. 19087-19898 USA, 2009), and employed automated liquid handlers toconduct serial dilutions and liquid transfers. The medium employed forthe MIC assay was Mueller Hinton II Broth (MHB II—Becton Dickinson,Sparks, Md.; Cat No 212322; Lot 9044411). S. aureus ATCC 29213 served asthe quality control strain, and linezolid was utilized as the qualitycontrol antibiotic to validate the assay. NCL812 and linezolid were bothdissolved in 100% DMSO before addition to the growth medium.

Macromolecular Synthesis Assays Bacteria and Growth Conditions

The effect of NCL812 on whole cell DNA, RNA, cell wall, protein andlipid synthesis was investigated using S. aureus ATCC 29213. Cells weregrown at 35° C. overnight on Trypticase Soy agar. A colony from theplate was used to inoculate 10 ml of Mueller Hinton broth II (MHBII),and the culture was grown to early exponential growth phase (OD600=0.2to 0.3) while incubating in a shaker at 35° C. and 200 rpm.

DNA, RNA, and Protein Synthesis

When cells reached early exponential phase, 100 μl of culture was addedto triplicate wells containing various concentrations of test compoundor control antibiotics (5 μl) at 20× the final concentration in 100%DMSO. A 5% DMSO treated culture served as the “no drug” control for allexperiments. Cells were added in MHBII at 105% to account for the volumeof drug added to each reaction or in M9 minimal medium for proteinsynthesis reactions. Following 15 min incubation at room temperature,either [3H] thymidine (DNA synthesis), [3H] uridine (RNA synthesis) or[3H] leucine (protein synthesis) was added at 0.5-1.0 μCi per reaction,depending on the experiment. Reactions were allowed to proceed at roomtemperature for 15-30 min and then stopped by adding 12 μl of cold 5%trichloroacetic acid (TCA) or 5% TCA/2% casamino acids (proteinsynthesis only). Reactions were incubated on ice for 30 min and the TCAprecipitated material was collected on a 25 mm GF/A filter. Afterwashing three times with 5 ml of cold 5% TCA, the filters were rinsedtwo times with 5 ml 100% ethanol, allowed to dry, and then counted usinga Beckman LS3801 liquid scintillation counter.

Cell Wall Synthesis

Bacterial cells in early exponential growth phase were transferred to M9minimal medium and added to 1.5 ml eppendorf tubes (100 μl/tube)containing various concentrations of test compound or controlantibiotics (5 μl) at 20× the final concentration in 100% DMSO asdescribed above. Following a 5 min incubation at 37° C.,[14C]N-acetylglucosamine (0.4 μCi/reaction) was added to each tube andincubated for 45 min in a 37° C. heating block. Reactions were stoppedthrough the addition of 100 μl of 8% SDS to each tube. Reactions werethen heated at 95° C. for 30 min in a heating block, cooled, brieflycentrifuged, and spotted onto pre-wet HA filters (0.45 μM). Afterwashing three times with 5 ml of 0.1% SDS, the filters were rinsed twotimes with 5 ml of deionized water, allowed to dry, and then countedusing a Beckman LS3801 liquid scintillation counter.

Lipid Synthesis

Bacterial cells were grown to early exponential growth phase in MHBIIbroth and added to 1.5 ml eppendorf tubes (in triplicate) containingvarious concentrations of test compound or control antibiotics asdescribed above. Following a 5 min incubation at room temperature,[3H]glycerol was added at 0.5 μCi per reaction.

Reactions were allowed to proceed at room temperature for 15 min andthen stopped through the addition of 375 μl chloroform/methanol (1:2)followed by vortexing for 20 seconds after each addition. Chloroform(125 μl) was then added to each reaction, vortexed, followed by theaddition of 125 μl dH2O and vortexing. Reactions were centrifuged at13,000 rpm for 10 min, and then 150 μl of the organic phase wastransferred to a scintillation vial and allowed to dry in a fume hoodfor at least 1 hr. Samples were then counted via liquid scintillationcounting.

Results

Susceptibility testing was conducted with NCL812 and S. aureus ATCC29213 to determine the concentrations of drug needed in themacromolecular synthesis assays.

FIG. 5 shows that the MIC for NCL812 was 4 μg/mL, while the qualitycontrol agent linezolid was within the CLSI-established quality controlrange (Clinical and Laboratory Standards Institute. PerformanceStandards for Antimicrobial Susceptibility Testing; NineteenthInformational Supplement. CLSI document M100-S20 [ISBN 1-56238-716-2].Clinical and Laboratory Standards Institute, 940 West Valley Road, Suite1400, Wayne, Pa. 19087-1898 USA, 2010). Precipitation of NCL812 wasobserved at ≧8 μg/mL in plates that were prepared in an identicalfashion, but did not receive an inoculum of S. aureus. Macromolecularsynthesis inhibition studies were performed using concentrations ofNCL812 that were equivalent to 0, 0.25, 0.5, 1, 2, 4 or 8-fold the MICvalue (4 μg/ml) for S. aureus ATCC 29213 (FIGS. 6-11).

FIG. 6 shows the effect of NCL812 on DNA synthesis. NCL812 demonstratedno inhibition at 0.25-fold the MIC, 40% inhibition at 0.5-fold, andapproximately 95% inhibition at the MIC. This is compared to the controlciprofloxacin which showed approximately 51% at 8-fold the MIC (0.5μg/ml).

The results for NCL812 inhibition of RNA synthesis were very similar tothe DNA synthesis study, with rifampicin serving as the positive control(FIG. 7). It should be noted that precipitation was observed at 4 to8-fold the MIC in the Mueller Hinton broth II utilized in the DNA andRNA synthesis assays.

Protein synthesis was inhibited in a dose-dependent manner at 0.25, 0.5,and 1-fold the MIC value of NCL812 showing up to 97% inhibition at theMIC (FIG. 8). Linezolid demonstrated approximately 61% inhibition ofprotein synthesis at 8-fold the MIC (2 μg/ml). Precipitation of NCL812occurred at 4 and 8-fold the MIC in the protein synthesis assay.

In FIG. 9, NCL812 also showed a somewhat dose-dependent inhibition ofcell wall synthesis, though there was a large increase in inhibitionfrom 1 to 2-fold the MIC. However, inhibition dropped to approximately68% and 52% at 4-fold and 8-fold the MIC, respectively. Precipitation ofNCL812 occurred at 2, 4, and 8-fold the MIC in the M9 minimal mediumused for the cell wall synthesis assay, and that is the likely cause ofthe decline in inhibition. In comparison, the positive controlvancomycin showed 96% inhibition at 8-fold the MIC (2 μg/ml).

NCL812 demonstrated a similar inhibition profile against lipid synthesisas that shown for DNA and RNA synthesis, reaching approximately 90%inhibition at the MIC (FIG. 10). The positive control inhibitorcerulenin demonstrated 72% inhibition at 8-fold the MIC (32 μg/ml).

FIG. 11 represents a composite of all five macromolecular synthesisreactions. It can be observed that the inhibition curves were similarfor each pathway, suggesting a global inhibition of several pathwayssimultaneously by NCL812. It is possible that NCL812 targets the cellmembrane, causing leakage of essential ions and/or metabolites, therebyleading to a global shutdown of the cell synthesis pathways.

In summary, NCL812 inhibited DNA, RNA, protein, cell wall, and lipidpathways in a growing culture of S. aureus. Though some instances ofdose-dependent inhibition of pathways was observed, all fivemacromolecular synthesis reactions were similarly sensitive to NCL812.

Example 3

Effect of NCL812 on ATP Release from Staphylococcus aureus

Materials and Methods Test Compounds

The test compound NCL812 was shipped under conditions of ambienttemperature and then stored at 2-8° C. until assayed. Stock solutionswere made by dissolving NCL812 dry powder in 100% DMSO to aconcentration of 1,600 μg/ml. The comparator agent was polymyxin B(Sigma, P-4932 (Lot 044K11905)).

Test Organism

S. aureus ATCC 29213 was originally acquired from the American TypeCulture Collection (Manassas, Va.).

ATP Release Assay

The CellTiter-Glo Luminescent Cell Viability Assay (Promega) wasutilized to measure the leakage of ATP from bacteria. Cultures weregrown to early exponential phase (0.2-0.3 optical density units at 600nm) in Mueller-Hinton Broth II and then treated with seven differentconcentrations of either NCL812 or polymyxin B (positive control)utilizing the MIC for each compound as a guide (0, 0.25, 0.5, 1, 2, 3,4, or 8-fold the MIC). The negative control received 2% DMSO, whichrepresented the final DMSO concentration in each assay. After a 30 minexposure to drug, cells were sedimented by centrifugation and thesupernatant was analyzed for the presence of ATP. Results were expressedas ATP concentration released to the medium (μM).

Results

The MIC for NCL812 has been previously determined to be 4 μg/ml. The ATPrelease assay is conducted by growing S. aureus to exponential phase andthen adding the antibiotic at multiples of the MIC in an effort todetect a dose-dependent response.

As shown in FIG. 12, the positive control polymyxin B released ATP fromS. aureus cells in a dose-dependent fashion with maximal release ofapproximately 0.34 μM ATP at 8-fold the MIC (256 μg/ml). ATP release inthe presence of NCL812 was dose-dependent at 0.5-1 fold the MIC,resulting in maximal release (0.33 μM) observed at the MIC (4 μg/ml).ATP release actually decreased thereafter at 2 to 8-fold the MIC. Itshould be noted that in previous studies, precipitation of NCL812 wasobserved at 4 to 8-fold the MIC in Mueller Hinton broth II.

In summary, NCL812 demonstrated dose-dependent release of ATP fromactively growing S. aureus cells. ATP release from the cells into thegrowth medium reached maximum levels at the MIC value, and this wasfollowed by a decrease in ATP release at higher doses. The dataindicated that NCL812 may interact with the cell membrane of S. aureus,causing leakage of vital metabolites such as ATP.

Example 4

In vitro antibacterial activity of NCL812 against methicillin-resistantand methicillin-susceptible Staphylococcus aureus

Materials and Methods Antimicrobial Agents

Aliquots of stock solution of NCL812 (25.6-mg/ml) was prepared indimethyl sulfoxide, stored at −80° C. and defrosted immediately beforeuse. Ampicillin stock was obtained from Sigma-Aldrich (Australia).Antimicrobial discs were obtained from Thermo Fisher Scientific(Australia).

Microorganisms

Twenty nine clinical isolates of MRSA were obtained (FIG. 13), alongwith S. aureus control organism ATCC 49775. Isolate identification wasconfirmed by conventional phenotypic methodologies, including the slidecoagulase test, Vogues-Proskauer test, polymyxin B sensitivity(300-units), and Staphytect Plus Protein A latex slide agglutination(Thermo Fisher Scientific Australia). Bacteria were stored at −80° C. in40% glycerol broth and routinely grown from stock on sheep blood agar(SBA) incubated at 37° C. In subsequent experiments, only fresh cultures<24-h were used.

Isolate Resistotyping

Antibiotic-susceptibility profiling of the isolate collection wasundertaken using Kirby-Bauer disc diffusion, as recommended by theClinical and Laboratory Standards Institute (CLSI) on Mueller-Hintonagar. Isolates were grown overnight on SBA at 37° C. Colonies weresuspended in physiological saline. Turbidity was adjusted to a 0.5McFarland standard and suspensions were spread over the medium.Antibiotic discs were transferred onto the inoculated medium andanalysed after 24-h incubation at 37° C. Isolates labelled as MRSA thatwere not β-lactam-resistant on the basis of the Kirby-Bauer test weregrown from stock on plate count agar supplemented with 5-μg/mlampicillin and subject to repeat testing, as Penicillin BindingProtein2a expression can be induced by exposure to β-lactamantimicrobials.

Molecular Detection of the Protein A and mecA Genes to Confirm MRSAStatus

Isolate identities were confirmed genotypically using a novel, duplexconventional polymerase chain reaction (PCR) test targeting the spa(protein A) and mecA (methicillin resistance) genes. In addition, theisolates were tested in a mecA and spa Sybr green real-time PCR.Approximately ten colonies of each overnight bacterial subculture wassuspended in 1× phosphate buffered saline (pH 7.4) and vortexed.Isolates were subject to DNA extraction using the QIAamp® DNA Mini Kit(Qiagen, Australia) following the manufacturers protocols. Template DNAwas eluted in 50-μl of elution buffer and either used directly in PCR,or stored at −20° C. prior to DNA amplification using the spa forward(5′-TGATACAGTAAATGACATTG-3′) and reverse (5′-TTCTTATCAACAACAAGTTC-3′)primers and mecA forward (5′-TTCGTGTCTTTTAATAAGTGAGG-3′) and reverse(5′-ATGAAGTGGTAAATGGTAATATCG-3′) primers (Invitrogen, Australia).Conventional PCR amplification was performed in a 20-μl volumecontaining 10-μl HotStarTaq Plus Master Mix (Qiagen, Australia), 0.5-μMof each spa primer, 0.2-μM of each mecA primer, and 3-μl of extractedDNA. An automated thermal cycler (T100 Thermal Cycler, Bio-Rad) was usedfor PCR amplification of the spa and mecA genes.

TABLE 1 PCR and RT PCR reaction conditions Temperature Time Number of (°C.) (seconds) cycles PCR stage Enzyme activation 95 300 1 Amplification:Denaturation 94 30 Annealing 50 30 38 Extension 72 38 Cooling 20 ∞ 1Real-time PCR stage Enzyme activation 95 300 1 Amplification:Denaturation 95 15 40 50 20 Annealing 70 40 Single acquisition 95 5 1Melting curve 55 20 95 0 Continuous acquisition Cooling 40 30 1

The mecA and spa amplified products of 325- and 120-bp, respectively,were detected by GelRed staining followed by electrophoresis in 2%agarose gels.

Minimum Inhibitory Concentration Testing

The in vitro activities of NCL812, and ampicillin as a positive control,were determined by broth microdilution as recommended by the CLSI incation-adjusted Mueller-Hinton II broth. Microtiter plates containingtwo-fold dilutions of each antimicrobial agent were inoculated with˜10⁵-CFU/ml of each isolate in a 100-μl final volume. Plates wereincubated for 24-h at 37° C. Turbidity (absorbance at OD₆₀₀) wasmeasured using a Bio-Rad Benchmark Plus microplate spectrophotometer inMicroplate Manager® version 5.2.1 (Bio-Rad). Minimum inhibitoryconcentration (MIC) endpoints were defined as the lowest antimicrobialconcentration assessed by the spectrophotometer that inhibited bacterialgrowth. ATCC 49775 was included in the isolate collection as a controlorganism using breakpoints defined by the CLSI. The MIC₅₀, MIC₉₀(concentrations that inhibited growth of the lower 50% and 90% of totalorganisms, respectively), and MIC range (minimum and maximum) werecalculated to profile the antimicrobial susceptibility of the isolatecollection.

Bactericidal Activity

The bactericidal activity of NCL812 was established by determination ofthe minimum bactericidal concentration (MBC) and time-kill analysesusing CLSI guidelines. The MBC was defined as the lowest drugconcentration at which 99.95% of the original inoculum was eliminated.

Time-kill assays for ATCC 49775 were performed in cation-adjustedMueller-Hinton II broth in Microtiter plates and again in 10-ml volumesfor macrodilution assays at antimicrobial concentrations equivalent to1× and 4× the MIC. Bactericidal activity in macrodilution assays wasidentified as a 3-log₁₀ decrease from the initial inoculum size.Bacteria were cultured overnight at 37° C. on SBA. Colonies weresuspended in broth and the turbidity was adjusted to a 0.5 McFarlandstandard to obtain a bacterial suspension of ˜10⁵-CFU/ml. Bacterialsuspensions were incubated at 37° C. with shaking. Aliquots were removedat 0-, 1-, 2-, 4-, 8-, 12-, and 24-h after antimicrobial addition,diluted, plated onto SBA and incubated for 48-h at 37° C. for viablecount determination. Turbidimetric growth curves for S. aureus wereobtained for Microtiter plate assays by monitoring optical densitychanges using a Bio-Rad Benchmark Plus microplate spectrophotometer at600 nm. Optical densities were measured at 0-, 1-, 2-, 4-, 8-, 12, and24-h after antimicrobial addition.

Statistical Methodology

Microbiological data was interpreted using CLSI guidelines. Data wasexamined using the student's t-test, Fisher's exact test, analysis ofvariance, and a generalized linear model for tests of between-subjectseffects where appropriate. Differences were considered significant atthe 0.05 level in IBM SPSS® version 19.0 (University of Adelaide).

Results

Confirmation of Staphylococcus aureus Identity and mecA Status

Latex agglutination tests confirmed that all 30 isolates were protein Apositive. The isolates tested positive for coagulase activity usingslide agglutination. Voges-Proskauer and polymyxin B resistance testsconfirmed that all isolates were S. aureus except for a singlemethicillin-susceptible isolate; MSSA DE-25 (FIG. 14). Based on spa genePCR amplification, this isolate was not identified as a S. aureusisolate despite testing positive in the protein A latex agglutinationand slide coagulase tests. This canine-origin Staphylococcus spp. wasidentified as Staphylococcus pseudintermedius based on biochemicalcharacteristics. The mecA conventional and real-time PCR resultsconfirmed that 66.66% of the isolates were classified asmethicillin-resistant on the basis of possession of the mecA gene. Therewere no significant differences between the ability of conventional andreal-time PCR to detect the mecA gene (P>0.05).

Staphylococcus aureus Antimicrobial Susceptibility Profiles

Antimicrobial susceptibility assays revealed that HA-MRSA isolates hadthe highest mean prevalence of resistance to multiple antimicrobialclasses (P<0.000). CA-MRSA isolates were next most resistant (P<0.007),followed by methicillin-susceptible staphylococci (P<0.037) (FIG. 15).Oxacillin resistance was expressed in only 80.00% and 10.00% of HA-MRSAand CA-MRSA isolates, respectively. Cefotetan resistance was expressedin 80.00% and 20.00% of HA-MRSA and CA-MRSA isolates, respectively.Although oxacillin and cefotetan did not significantly differ in theirability to detect MRSA (P>0.05), detection was significantly improvedwhen using the mecA PCR when compared to disc diffusion (P<0.013). Themajority of HA-MRSA isolates expressed resistance toamoxicillin-clavulanic acid, cefotetan, cephalexin, clindamycin,erythromycin, oxacillin, and penicillin-G, whereas the majority ofCA-MRSA isolates were resistant to only clindamycin, erythromycin, andpenicillin-G. None of the isolates tested were vancomycin resistant.Overall, the most prevalent resistance phenotypes were penicillin-G(83.33%), erythromycin (73.33%), and clindamycin (43.33%), whilst onlysingle isolates (3.33%) were resistant to trimethoprim-sulfamethoxazoleand rifampicin.

Mec Gene Complex Interactions

All MRSA isolates belonging to mec gene complex A expressed resistanceto both oxacillin and cefotetan (FIG. 16). However, only 20% of mec genecomplex B MRSA isolates were phenotypically resistant to theseantimicrobials. Of the MRSA isolates belonging to mec gene complex C2,only a single isolate expressed methicillin resistance to oxacillin andonly two isolates expressed resistance to cefotetan. Unclassified MRSAisolates expressed full resistance to oxacillin and cefotetan.

Melting point peaks for the mecA real-time PCR negative derivative plot−dF/dT differed between mec gene complex (P<0.003) (FIG. 17). Onaverage, mec gene complex B and unclassified isolates demonstratedhigher melting point peaks than other SCCmec types (P<0.012).

Physical Properties and MIC of NCL812

Initial tests of NCL812 showed that it was soluble in DMSO, but produceda cloudy solution when dissolved in cation-adjusted Mueller-Hinton IIbroth (CAMHB) (FIG. 18). In initial testing, NCL812 was found to haveconsistent MIC values (FIG. 18).

In Vitro Antibacterial Activities: Minimum Inhibitory Concentrations

MIC₅₀ and MIC₉₀ values for compound NCL812 (4- and 4-8-μg/ml) are shownin FIG. 19. MIC values differed by S. aureus classification(susceptible, HA- or CA-MRSA) (P<0.005). In many cases, NCL812 hadsignificantly increased activity against CA-MRSA andmethicillin-susceptible staphylococci by one dilution when compared toHA-MRSA (P<0.002 and P<0.020, respectively), however there were nosignificant differences between MIC values for methicillin-susceptiblestaphylococci and CA-MRSA (P>0.05). Ampicillin MIC values were withinthe normal range expected on the basis of CLSI guidelines.

In Vitro Antibacterial Activities: Minimum Bactericidal Concentrations

The MBCs determined from NCL812 were equivalent to the MIC for 93.33%and 83.33% of S. aureus isolates, respectively (FIG. 19). In allremaining cases, MBCs were one dilution higher. For NCL812, MBCs rangedfrom 2-8-μg/ml and 4-16-μg/ml, respectively.

Time-Kill Studies

In comparison to the turbidimetric growth curve of ATCC 49775, novisible bacterial growth was observed when ATCC 49775 was inoculatedinto cation-adjusted Mueller Hinton II broth supplemented with NCL812 at1× and 4× the MIC in microdilution assays (P<0.033 and P<0.038,respectively) (FIG. 20).

When analysed in 10-ml macrodilution assays, broth supplemented withantimicrobials at 1× and 4× the MIC and inoculated with ATCC 49775displayed significantly reduced viable counts for both NCL812 whencompared to the growth control (0.000<P<0.008) (FIG. 21). Additionally,the time-kill profiles of NCL812 did not significantly differ (P>0.05).Both antimicrobials remained bactericidal until approximately 8-12-hafter antimicrobial addition, where bacterial regrowth was observed.Considerable variation in the killing activity of NCL812 was observedfrom 8-24-h. Although NCL812 was no longer bactericidal by 24-h, viablecounts observed at 1× the MIC remained significantly lower than thoseobtained from unsupplemented broth (P<0.046).

In summary, the example set out above demonstrates bactericidal activityby NCL812 against both methicillin-susceptible staphylococci and MRSA.MIC and MBC values were consistently low across the selection ofisolates (MIC_(range) 2-8-μg/ml). NCL812 retained good in vitroantimicrobial activity against common, multidrug-resistant MRSAisolates, including the epidemic UK EMRSA-15, EMRSA-16, and EMRSA-17,Irish EMRSA-1, AUS EMRSA-3, NY/JAPAN HA-MRSA, and predominant CA-MRSAclones. NCL812 was also active against one S. pseudintermedius isolatethat was originally identified as a S. aureus strain.

Preliminary testing suggests that NCL812 targets the S. aureus cellmembrane, causing dose-dependent release of vital metabolites such asadenosine-5′-triphosphate. Disruption of the bacterial membrane bilayeror proteins that are integral to membrane function in bacteria is atarget for numerous large antimicrobials which are ubiquitous in nature;including glycolipids, lipopeptides, lipoproteins, fatty acids, neutrallipids, phospholipids, and biosurfactants. Although NCL812 is a lowmolecular mass (≦500-Da) synthetic compound, it does appear to exertbactericidal activity in a similar manner to other antimicrobials whichtarget the Gram-positive cell membrane, including the high molecularweight cyclic lipodepsipeptide antimicrobial agent daptomycin, or thelow molecular mass quinolone-derived HT61, whose chemical structure isnot currently available. Many of these lipophilic antibacterial agentsare not effective against Gram-negative microorganisms due to thepresence of the outer lipid bilayer membrane, which contains narrowporin channels reducing the net penetration of some compounds into thecell.

The insolubility of NCL812 at even low concentrations in microbiologicalmedia may reflect the amphipathic and oligomeric nature of thisantimicrobial and suggests that the real MIC may be much lower thanobserved, as it is likely that it is only NCL812 in solution that isbiologically active. In time-kill studies, NCL812 exerted rapid in vitrobactericidal activity against ATCC 49775.

Importantly, the apparent short in vitro half-life of this antimicrobialresulted in bacterial regrowth observed at 12-h after antimicrobialaddition. This suggests that if a viable bacterial population survivesthe initial exposure to NCL812 prior to antimicrobial inactivation,bacterial regrowth will occur. The development of resistance to NCL812in these studies was ruled out as test bacteria remained susceptible toNCL812 following harvesting, washing and MIC testing. Whilst theapparent short in vitro half-life of NCL812 may be a desirablecharacteristic for future in vivo application, it does suggest thatNCL812 may need to be administered every 8-h in future in vivo safetyand efficacy experiments to maintain adequate systemic concentrations,though it would appear from the time-kill profile that the NCL compoundseries are concentration-dependent rather than time-dependentantimicrobials.

To overcome the methicillin-susceptible MRSA phenotype, extending discdiffusion incubation time from 24- to 48-h compensates for the slowderepression of the mecR gene. Although the effects of longer incubationwere not examined, and the small sample size of MRSA isolates preventedfurther investigation into mec complex interactions; genetic techniqueswere of significantly improved sensitivity when compared to phenotypicmethods for confirmation of the mecA status of the isolates in thisstudy. Although genetic techniques are not always employed as a routinemethod for detecting MRSA, real-time PCR identification of the presenceof the mecA gene in a Staphylococcus spp. isolate remains the diagnosticgold standard.

Example 5

In vitro pharmacodynamics of a new antimicrobial agent for Streptococcuspneumoniae.

Materials and Methods Pneumococcal Antimicrobial SusceptibilityPneumococcal Strains and Growth Conditions

Twenty pneumococcal isolates comprised of eight characterised laboratorystrains and 12 clinical isolates were obtained.

TABLE 2 Characterisation of Isolates Tested Infectious Dose StrainSerotype Reference (ID₅₀) by IP D39 2 Avery et al. 2010 Nature 10²*(NCTC Reviews Microbiology 7466) 8: 260-271 A66.1 3 Francis et al. 2001.Infect 8 × 10³  Immun. 69: 3350-2358 (10, 98) EF3030 19F Briles et al.2003 J. Infec. ≧10⁵* Diseases. 188: 339-348 L82016   6B Briles et al.2000 Infect Immun. ≧10⁵*   68: 796-800 P9   6A This study 10⁴*   P21 3This study ≦10¹* TIGR4 4 Tettlelin et al. 2001 Science 10⁴* 293: 498-506WU2 3 Briles et al. 1981 J. J Exp Med.  5 × 10¹²* 153: 694-705 WCH16  6A This study 5 × 10⁴* WCH43 4 This study 10²* WCH46 4 This study 10⁴*WCH57 8 This study 10⁴* WCH77 5 This study 10⁴* WCH86 4 This study 10⁴*WCH89 7 This study ≧10⁵* WCH92 4 This study ≦10⁴* WCH137 6A This studyNot determined to date WCH158 19F This study 10⁵* WCH184 19F This study10⁸ (14) WCH211 11  This study 5 × 10⁶*

The National Collection of Type Cultures (NCTC) control strain D39 wasused as a growth control for all MIC and MBC assays. D39 was laterdesignated for kill kinetics, point of resistance assays andtransmission electron microscopy (TEM) studies as it is a welldocumented laboratory strain with a defined in vivo pathogenesis thatdisplayed consistent NCL812 MICs and MBCs.

For all in vitro assays, fresh pneumococcal isolates were grownovernight (O/N) on horse blood agar (HBA) plates (39 g/L Columbia bloodagar base [Oxoid] 5% [v/v] defribinated horse blood [Oxoid] at 37° C.with 5% supplemented CO₂. Mueller-Hinton blood agar with 5% defibrinatedsheep blood (MHSBA Roseworthy Media and Blood Service) was used for diskdiffusion analysis as directed by Clinical Laboratory StandardsInstitute (CLSI) standards. Pneumococci were routinely grown in brothconsisting of 4% lysed horse blood (LHB) with Cation Adjusted MuellerHinton Broth (CAHMB, [Difco]) at 37° C. with 5% supplemented CO₂. Horseserum broth (HSB, 10% (v/v) donor horse serum in nutrient broth [10 g/Lpeptone, 10 g/L Lab Lemco (Oxiod) and 5 g/L NaCl]) was also used in someMIC assays. Isolates were stored in HSB at −80° C.

Antibiotic Stocks and Reagents

NCL812 was provided in dry powder form by Neoculi. A total of 256 mg wasdispensed into 10 ml of 100% dimethyl sulphoxide (DMSO) to make a stockof 25.6 mg/ml, which was then diluted 1:100 in CAHMB to make a finalworking stock of 256 μg/ml. Ampicillin dry powder was from Sigma A0166.The original 25.6 mg/ml stock was diluted in saline 1:100, 1:4, 1:20 andfinally 1:16 in CAMHB to make a final working stock of 0.18 μg/ml.Erythromycin was from Sigma E077 and choline chloride was from RocheDiagnostics. Twenty micro litres of 0.05 μg/ml erythromycin was diluted1:25 in 4.980 mls of CAMHB to give a final working stock of 0.2 μg/ml.Choline chloride (0.5%) was added to 4% LHB:CAMHB for specific killkinetic assays.

Defining Antimicrobial Susceptibility of Pneumococcal Isolates

Isolate susceptibility to 12 different antimicrobials (FIG. 22) weredetermined by CLSI and European Committee on AntimicrobialSusceptibility Testing (EUCAST) methods. Antimicrobials were selectedbased upon the CLSI and EUCAST guidelines. Zone diameters forantimicrobials other than Ciprofloxacin for S. pneumoniae weredetermined by CLSI standards; whilst Zone diameters for Ciprofloxacinantimicrobial susceptibility to S. pneumoniae were determined by EUCAST.

TABLE 3 Interpretive Standards for Zone Diameters Interpretive Standardsfor Zone Diameters (mm) Antibiotic Class Antimicrobial (μg) Resistant(R) Intermediate (I) Sensitive β-lactam Oxacillin (1 μg)° ≦20 ≦20 ≧20Ampicillin (10 μg)° ≦20 ≦20 ≧20 Amoxicillin-clavulanate ≦20 ≦20 ≧20(20/10 μg)° Fluoroquinolone Ciprofloxacin (5 μg)* ≦22 ≦22 ≧22 Folatepathway Trimethoprim- ≦15 16-18 ≧19 inhibitor sulphamethoxazole(1.25/23.75 μg)° Glycopeptide Vancomycin (30 μg)° — — ≦17 LincosamideClindamycin (2 μg)° ≦15 16-18 ≧19 Macrolide Erythromycin (15 μg)° ≦1516-20 ≧21 Clarithromycin (15 μg)° ≦16 17-20 ≧21 Phenicol Chloramphenicol(30 μg)° ≦20 — ≧21 Rifamycin Rifampin (5 μg)° ≦16 17-18 ≧19 TetracyclineTetracycline (30 μg)° ≦18 19-22 ≧23

Standardised bacterial suspensions were spread onto MHSBA using asterile cotton swab. [Bacterial suspensions from of Streptococcuspneumoniae were standardised to an OD_(600 nm) between 0.08 and 0.1using a spectrophotometer and then diluted 1:20. Bacterial colonies weretaken from an O/N horse blood agar plate. To ensure the purity of the1:20 bacterial suspension, 50 μL was spread plated onto horse blood agarand incubated O/N at 37° C. with 5% CO₂. The CFU was calculated andcompared to the initial plate counts.] Antibiotic disks (Sigma) wereplaced using a disk dispenser (Oxoid) according to CLSI standards. MHSBAplates were incubated for 16 hrs-24 hrs at 37° C. in 5% CO₂. Zones ofcomplete inhibition were measured in triplicate to the nearestmillimetre using a ruler on natural light-reflected growth, and the modewas represented as the diameter for each isolate. Pneumococcal isolateswere categorised as sensitive, intermediate (I) or resistant (R) by CLSIstandards and quality control (QC) ranges (FIG. 22).

Determination of NCL812 MIC50, MIC90, MIC Range and MBC50, MBC90, MBCRange

MICs for NCL812 for all isolates listed in Table 2 were determined bymeasuring optical density at 600 nm (OD600 nm) (Spectramaxspectrophotometer, Molecular Devices Corporation) as an indicator ofbacterial growth using 96-well microtitre trays after incubation for 24hrs at 37° C. in 5% CO₂. [Micro-broth dilutions and 96-well trays areprepared by the following method: 90 μL of 4% LHB: CAMHB is aliquottedinto all wells using a multichannel pipette. 90 μl of workingantimicrobial stocks were no serial diluted down the tray by a 1:2dilution. Negative broth controls and dilution control were taken intoaccount when planning the set up of a 96-well tray.] 10 μL of bacterialsuspension was then added to the appropriate wells in the 96 well tray.Appropriate positive (no antimicrobial), negative (no antimicrobial orbacteria) and negative dilution (a serial dilution control ofantimicrobial and broth) controls were included in each assay. MBC andplate counts for kill kinetic assays were determined by aliquotting 20μL from each well of the 96-well microtitre tray onto HBA, andincubating at 37° C. with 5% CO2. The MBC was determined by a 99.95%inhibition of S. pneumoniae, taking into account the dilution factor.MICs and MBCs were determined in quadruplicate and the mode was taken asthe representative value. The MIC50, MIC90 and MIC range and MBC50,MBC90 and MBC range were determined according to CLSI standards. TheMIC50 and MIC90, or MBC50 and MBC90, are defined by the lowestconcentrations which, when all the MICs and MBCs of the isolates arearranged from lowest to highest, inhibited the 50th and 90th percentileof the total amount of isolates, respectively.

Micro-Broth Dilution Time Kill Studies with NCL812 Using Strain D39

Bacterial suspensions were added in triplicate to a 96-well microtitretray containing NCL812 with a starting concentration of 128 μg/ml andserially diluted 1:2 sequentially to a concentration of 0.25 μg/ml.Negative dilution controls were subtracted from the median growth valueto obtain a suitable indicator of overall bacterial production. The96-well tray was incubated at 37° C. in 5% CO₂ and read every 2 hrs forthe first 12 hrs followed by final reads at 24 and 48 hrs at 600 nm. Tofurther supplement this data, a separate experiment in which a 96-welltray was read automatically at half hourly intervals using aspectrophotometer (Spectramax spectrophotometer, Molecular DevicesCorporation) for 14 hrs was performed to confirm the trends in growthcurves observed from original micro-broth dilution studies.

MBC Time Kill Studies with NCL812 Using Strain D39

MBC kill kinetics assays involved the preparation of three 96-wellmicrotitre trays. At specific time points, aliquots obtained from thesetrays provided viable counts following incubation at 37° C. in 5% CO₂ onHBA, and the MBC was determined after 24 hrs of growth.

Macro-Broth Dilution Time Kill Studies of D39 with NCL812

Bacterial suspensions and working antibiotic stocks were prepared asdescribed above. [For preparing macro-broth dilutions, 20 ml tubes werefilled each with 9 mls of 4% LHB:CAMHB. 9 mls of a working antimicrobialstock was diluted 1:2 when added to one of the tubes, and then serialdiluted down from a high to low concentration of antimicrobial. 1 ml ofS. pneumoniae bacterial suspension was added to the appropriate tubes,including the positive control. Tubes were incubated at 37° C. with 5%CO₂ with gentle manual tilting of the tubes treated with NCL812 every 10mins for the first 12 hrs. At every 2-3 hrs during the first 12 hrs ofgrowth and then at 24 hrs and 48 hrs, 50 μL of each bacterial suspensionwas spread plated onto HBA and incubated at 37° C. with 5% CO₂ for 16-24hrs.]

TABLE 4 Concentration of antimicrobials Serial dilution NCL-812 (μg/ml)Ampicillin (μg/ml) 1 128 0.09 2 64 0.045 3 32 0.023 4 16 0.011 5 80.0065 6 4 7 2

Cultures were incubated at 37° C. in 5% CO₂ with gentle manual tiltingevery 10 mins for the first 12 hrs. Viable counts from 50 μL aliquots ofeach concentration were read following incubation at 37° C. in 5% CO₂for 24 hrs. The pH of each sample was measured at specific time pointsusing pH indicator strips. Confluent growth was defined when more than1000 colonies were counted per plate. A bactericidal effect was definedas a 3-log 10-unit reduction (99.9%) of the original cell suspensiondetermined at 24 hrs for each concentration.

Point of Resistance Assay for NCL812

Macro-broth dilutions were prepared as above. Broth cultures of strainD39 (10 ml) were incubated in the presence of 2 μg/ml and 4 μg/ml ofNCL812, and 0.022 μg/ml of Ampicillin for 6 hrs at 37° C. in 5% CO₂.Samples were centrifuged at a relative centrifugal force (RCF) of101.45×g for 10 mins and washed in 50 mls of phosphate buffered saline(PBS) twice to remove any residual antimicrobial, and/or bacterial endproducts and media. Washed bacteria were resuspended and MICs wereperformed.

Effect of NCL812 on D39 Cell Membrane Ultra-Structure TransmissionElectron Microscopy

Morphological appearance and morphometric analysis of the cell membranewas determined using transmission electron microscopy (TEM). Bacterialsuspensions and 10 ml cultures of D39 were prepared as before. Sampleswere incubated at 37° C. in 5% CO₂ with gentle manual tilting of thecultures every 10 mins. Cultures were exposed to either 1 μg/ml, 4 μg/mlor 16 μg/ml of NCL812 and harvested at 6 or 12 hrs by centrifugation at101.45×g for 20 mins and washed twice in 50 mls of PBS. Critical timepoints for TEM work were determined by analysing trends in the growthcurves produced from the kill kinetics studies. Samples were resuspendedin PBS containing 20% glycerol and stored at −80° C. until required.Before fixation, 20% glycerol was removed by centrifugation and washingon ice three times in 50 mls of PBS.

Samples were fixed using modified protocols defined by a previous studyexamining cell wall ultrastructure of S. pneumoniae (Hammerschmidt, S.et al. 2005. Infect Immun 73:4653-4667). A lysine-acetate-basedformaldehyde-glutaraldehyde ruthenium red-osmium fixation procedureinvolved fixing the bacterial pellets with a cacodylate buffer solutioncontaining 2% formaldehyde, 2.5% glutaraldehyde, 0.075% ruthenium redand 0.075 M of lysine acetate for 1 hr. After washing with cacodylatebuffer containing 0.075% ruthenium red three times, a second fixation incacodylate buffer solution containing 2% formaldehyde, 2.5%glutaraldehyde and 0.075% ruthenium red was undertaken for 1.5 hrs.Cells were subsequently washed three times with cacodylate buffercontaining 0.075% ruthenium red and underwent a final fixation in 1%osmium tetroxide in cacodylate containing 0.075% ruthenium red for 1 hr.The samples were then washed three times in cacodylate buffer containing0.075% ruthenium red only.

Samples were washed and dehydrated using a graded series of ethanol (70,90, 95 and 100%) for 10-20 min, two times for each step. Samples wereinfiltrated using 50:50 LR White resin in 100% ethanol for 1 hr, andsubsequently washed with 100% LR White resin for 1 hr and left O/N in athird change of 100% LR white to ensure adequate infiltration of resin.The samples were then embedded in fresh LR White resin and incubated at50° C. for 48 hrs. Sections were cut to 1 μm using a glass knife,stained with Toluidene Blue and viewed under a light microcrope at 400×to identify the presence of stained pneumococci. At least fourultra-thin sections were then cut to 90 nm using a diamond knife andplaced on matrix grids, one section per grid. Ultra-thin sections werethen stained with uranyl acetate and lead citrate alternatively at 5 minintervals, followed by three washes with distilled water in-between eachexposure. Stained sections were then placed on grids and viewed between25000× and 130000× on a Philips CM100 Transmission Electron Microscope.Images were obtained at 130000× magnification and analysed usinganalySIS [Olympus Soft Imaging Systems].

Statistical Analysis

Statistical analyses were conducted using statistics program GraphPadPrism (5th ed, GraphPad Software Inc.) for Windows. For growth curves,data presented were the mean and standard error of mean (SEM)(represented as error bars) for each data point except for macro-brothdilution studies where multiple replicates could not be obtained due tothe high costs involved in this assay. Two tailed, unpaired t-tests wereperformed.

Results

Pharmacodynamics of NCL812 in S. pneumoniaeQuality Control Disk Diffusion Analysis for 20 S. pneumoniae Isolates

Although nine out of the 12 antimicrobials used for disk diffusionanalysis had established QC ranges by EUCAST, QC ranges were not definedfor amoxicillin-clavulanate, clarithromycin and clindamycin (Table 3).

WCH16 and WCH184 were both resistant to at least two antimicrobialswhereas EF3030 and WCH137 were intermediate and resistant totrimethoprim-sulphamethoxazole respectively (FIG. 22). The otherremaining 16 isolates were sensitive to all 12 antimicrobials.Sensitivity to ampicillin was confirmed for each isolate, enabling theuse of ampicillin as a positive control in later micro-broth dilutionassays (FIG. 22).

Solubility and Activity of NCL812 in Different Media

NCL812 was brought in 100% DMSO but developed turbidity when it wasfurther diluted into CAMHB or PBS.

TABLE 5 Visual analysis of NCL812 and ampicillin solubility NCL812Ampicillin Diluent CAMHB Turbid Transparent DMSO Transparent TransparentPBS Precipitate Transparent Media 4% LHB:CAMHB Turbid Transparent 10%horse serum-supplemented broth Precipitate Transparent

Growth of S. pneumoniae strain D39 in an MIC assay for NCL812 using 10%HSB (220 mls of horse serum is filtered to 10% in 180 mls of Lemconutrient broth) resulted in a threefold increase in the MIC for D39treated with NCL812 with a twofold increase for the positive ampicillincontrol.

TABLE 6 Difference in activity of NCL812 in different media. RelativeMIC with media type for D39 4% 10% horse serum- Antimicrobial LHB:CAMHBsupplemented broth Fold-increase NCL812 4 32 3 Ampicillin 0.023 0.09 2

There was no change in MIC for D39 with differing storage conditions ofpre-prepared 96-well microtitre trays.

TABLE 7 Storage conditions of prepared micro titre trays for micro brothdilution. Storage condition Antimicrobial −2° C. 4° C. NCL812 8 8Ampicillin 0.023 0.023

During macro-broth dilutions, the pH of the media did not changecompared to appropriate controls (FIG. 23).

Determination of S. pneumoniae In Vitro Susceptibility to NCL812

Determination of NCL812 MIC50, MIC90, MIC Range

NCL812 exhibited a MIC50 and MIC90 of 8 μg/ml and MIC range of 4-8 μg/mlwhen tested against all 20 strains. The MIC for ampicillin wascomparable to recent published findings using micro-broth dilution as anendpoint for antimicrobial resistance in pneumococcal isolates, thusconfirming the accuracy of MICs obtained for NCL812.

TABLE 8 MIC and MBC values for isolates treated with NCL812 andampicillin NCL812 (μg/ml) Ampicillin (μg/ml) MIC₅₀ 8 0.023 MIC₉₀ 8 0.023MIC Range 4-8 0.011-0.09 MBC₅₀ 8 0.023 MBC₉₀ 8 0.023 MBC Range 4-80.011-0.09

Determination of NCL812 MBC50, MBC90, MBC Range

Minimum bactericidal concentrations (MBC50, MBC90 and MBC rangerespectively) were determined for NCL812 and ampicillin for all twentyisolates.

TABLE 9 MIC and MBC values for NCL812 and ampicillin for eachpneumococcal isolate NCL812 Ampicillin MIC MBC MIC MBC D39 4 8 0.0230.023 EF3030 8 8 0.023 0.023 A66.1 8 8 0.045 0.045 TIGR4 4 8 0.023 0.023WU2 4 8 0.023 0.023 L82016 8 8 0.023 0.023 P9 8 8 0.023 0.023 P21 4 80.023 0.023 WCH158 4 8 0.023 0.023 WCH89 4 4 0.023 0.023 WCH57 8 8 0.0230.023 WCH77 4 8 0.023 0.023 WCH46 4 4 0.023 0.045 WCH86 4 8 0.023 0.023WCH137 4 8 0.023 0.023 WCH184 4 4 0.045 0.045 WCH16 8 autolysis 0.023Autolysis WCH43 4 8 0.023 0.023 WCH92 8 8 0.09 0.09 WCH211 4 8 0.0230.023Micro-Broth Dilution Time Kill Studies of D39 Treated with NCL812

D39 exposed to sub-inhibitory concentrations (≦2 μg/ml) of NCL812 grewsimilar to unexposed controls over a 48 hour period (FIG. 24). Higherconcentrations of NCL812 (≧16 μg/ml) resulted in no bacterial growth for48 hrs (FIG. 24). These growth characteristics were validated by amicro-broth kill kinetic study using a Spectramax spectrophotometer,which measured growth (represented as OD600) at half-hourly intervalsfor 14 hrs for NCL812 and ampicillin (FIGS. 25 and 26). The commencementof exponential growth for D39 treated with NCL812 is shown in FIG. 27.

The growth of D39 treated with NCL812 was compared to D39 treated withampicillin or erythromycin over 48 hrs (FIGS. 28 and 29). D39 treatedwith ampicillin exhibited similar growth to D39 exposed to NCL812 over48 hrs (FIG. 28). Erythromycin-treated D39 produced very differentgrowth curves from NCL812 where a larger difference in growth betweenconcentrations was observed (FIG. 29). The addition of 5% cholinechloride to the media over a 48 hour period resulted in no significantdifference in growth for NCL812 compared to positive and growth controls(FIGS. 30 and 31).

Point of Resistance Testing

D39 treated with ≦4 μg/ml NCL812 entered a log phase of growth at 6 hrs(FIG. 24), as shown in four independent experiments. The possibility ofantimicrobial resistance to NLC812 between 5 and 6 hrs was investigatedby determining further MICs on D39 exposed to 2 μg/ml NCL812, 4 μg/mlNCL812 and 0.0225 μg/ml ampicillin for 6 hrs. Results showed nosignificant increase in MIC for all samples of D39 exposed to NCL812compared to growth controls, and ampicillin

TABLE 10 MIC and MBC values of D39 exposed to 2 ug/ml or 4 ug/ml ofNCL812 for 6 hours MIC of D39 MBC of D39 following following Originalexposure to Original exposure to MIC of NCL812 MBC of NCL812 D39 for 6hrs. D39 for 6 hrs D39 exposed to 2 μg/ml 4 8 8 8 NCL812 D39 exposed to4 μg/ml 4 8 8 8 NCL812 D39 exposed 0.023 0.045 0.023 0.023 0.023 μg/mlAmpicillin D39 growth* 8 8 8 8 D39 growth2** 8 8 8 8 *D39 growthcontrol: S. pneumoniae strain D39 grown for 6 hrs in 4% LHB:CAMHB. **D39growth2 control: S. pneumoniae strain D39 on HBA O/N, resuspended insaline (0.1 OD₆₀₀) and diluted 1/20 in sterile saline.

Micro-Broth Dilutions by Measuring Relative MBC at Specific Time Points

Relative MBCs were determined at specific time intervals from usingbroth dilution assays incubated for 48 hrs for NCL812 and controlantimicrobials ampicillin and erythromycin (FIGS. 32 and 33). MICs ofampicillin (0.023 μg/ml) and erythromycin (0.00275 μg/ml) for D39 weresimilar in range to published findings for other pneumococcal isolates.A comparative difference in growth between NCL812, ampicillin, anderythromycin was observed (FIGS. 32 and 33). Ampicillin and erythromycindemonstrated a time-dependent reduction in bacteria. NCL812 exhibitedfast bactericidal action, evidenced by an approximate 3-fold decrease inMBC within 5 hrs. A consistent bactericidal concentration (8 μg/ml) wasmaintained for the full 48 hrs for NCL812.

Macro-Broth Dilution Time Kill Studies of D39 with NCL812

Viable counts for each time point were represented as a log 10 CFU/mlreduction for NCL812 (FIG. 34) and ampicillin (FIG. 35). Consistentconfluent growth (determined by a limit of 2×10⁴ CFU) was observed forunexposed controls and 2 μg/ml NCL812. Complete bactericidal activity(defined by a 3-log reduction in CFU) for 128 μg/ml of NCL812 wasobserved by a 4-log reduction of colony forming units (CFU) in 3 hrs andconcentrations between 16 μg/ml and 64 μg/ml NCL812 were effective ateliminating bacterial growth within 8 hrs (FIG. 34). NCL812 at 4 μg/mland 8 μg/ml appeared to be inactivated at 11 hrs post-exposure, asincreased growth of strain D39 after this time point was observed (FIG.34).

Transmission Electron Microscopy

Morphometric analysis revealed significant changes to the cell membranein strain D39 exposed to 16 μg/mL NCL812 for 6 hrs compared to growthcontrols. Samples treated with 4 μg/ml as well as 12 hr cultures werenot considered for morphemetric analysis due to the lack of bacterialcells available in each section. Treated samples possessed significantlythicker cell membranes (6.43±0.29 nm) compared to untreated samples(4.35±0.24 nm) (p<0.0001) (FIG. 36). The periplasmic space(intracellular space between the cell membrane and the cell wall) of D39treated with 16 μg/ml NCL812 was significantly wider (4.54±0.096 nm)compared to untreated samples (3.91±0.14 nm) (p<0.001) (FIG. 37).

TABLE 11 Mean cell membrane thickness and periplasmic space Treatment(16 μg/ml Unpaired Growth control NCL812 for 6 hrs) t-test Statisticaltest Mean ± SEM Mean ± SEM (P value) Cell membrane 4.35 ± 0.24 nm, 6.43± 0.29 nm, P < 0.0001 n = 12 n = 13 Periplasmic space 3.91 ± 0.14 nm,4.54 ± 0.096 nm, P < 0.001 n = 11 n = 11

In summary, NCL812 produced highly consistent MICs and equivalent MBCsfor the S. pneumoniae strain collection, confirming that it isbactericidal against this organism. In kill kinetics experiments, whichmeasured the relative MBC over a 48 hr period, a consistent bactericidaleffect was elicited in D39 after 6 hrs from initial exposure to NCL812.

This demonstration of bactericidal activity is the first to be observedin S. pneumoniae. This demonstrates that NCL812 is effective againstpneumococcal in vitro.

Competitive binding between components in blood, serum or brothdecreased the antimicrobial activity of NCL812. This was reflected inthe increase of MIC observed between different broth types and diluents.Following the completion of these studies, recent independent researchconfirmed precipitation of NCL812 in PBS and reported completesolubility in water containing 4% DMSO, following initial dilution in100% DMSO. A water-soluble NCL812 will greatly improve in vivobioavailability and negative interaction between blood or serumproteins.

Based on the findings of this study, NCL812 exhibits a mechanism ofaction against S. pneumoniae that is different from β-lactam ormacrolide classes, as it appears to exhibit concentration-dependentbactericidal activity as opposed to time-dependant qualities.Identifying the maximum pharmacokinetic serum concentration of NCL812 invivo will assist confirmation of its concentration-dependantpharmacodynamic activity. Furthermore, the addition of choline chlorideto the media confirmed that the mechanism of action for NCL812 is notassociated with the affinity to cell wall choline binding proteins, andtherefore may not be cell wall associated.

Morphometric analysis of the cell membrane and periplasmic space of D39treated with 16 μg/ml NCL812 for 6 hrs showed that the cell membrane andperiplasmic space was larger in treated samples, compared to controlsamples. The apparent increase in membrane size could be due to anaccumulation of electron dense intracellular material beneath the cellmembrane. The increase in the size of the periplasmic space may be havebeen due to disruption of the cell membrane, potentially bydepolarisation or ATP inhibition. The mechanism of action of NCL812 maynot be calcium-dependant as it appears that no competitive bindingbetween NCL812 and ruthenium red, a calcium channel inhibitor of lipidbilayers, was observed in electron micrographs.

In conclusion, this in vitro study has demonstrated that NCL812 has manydesirable characteristics as a fast-acting concentration-dependentbactericidal antimicrobial that appears to target the cell membrane ofS. pneumoniae. These characteristics are desirable to treat acutepneumococcal infections. As NCL812 may possess a mechanism of actionthat targets the cell membrane, it will act much more quickly thantime-dependent antimicrobials such as β-lactams and macrolides andpotentially could be more effective than other bactericidalconcentration-dependent antimicrobials such as fluoroquinolones whichhave intracellular targets.

Example 6

Characterization of methicillin-susceptible and methicillin-resistantisolates of Staphylococcus pseudintermedius from Australia andpreliminary in vitro efficacy of a new anti-staphylococcal compound

Materials and Methods

Sample Collection and Identification of Methicillin SusceptibleStaphylococcus pseudintermedius (MSSP) and Methicillin ResistantStaphylococcus pseudintermedius (MRSP)

A total of 23 Staphylococcus pseudintermedius isolates were obtainedfrom dogs (FIG. 38).

Ten methicillin susceptible and 13 methicillin resistant Staphylococcuspseudintermedius were collected for the study. Isolates werephenotypically classified as methicillin resistant on the basis of invitro resistance to oxacillin and genetically for the presence of mecAgene according to standard procedures.

Oxacillin and cefoxitin susceptibility testing using disk diffusiontechnique and Epsilometer testing were performed. Identification of mecAgene was performed using polymerase chain reaction (PCR)

CLSI disk diffusion susceptibility testing was performed on the 23Staphylococcus pseudintermedius isolates for the followingantimicrobials: penicillin, amoxicillin, erythromycin, gentamicin,clindamycin, ciprofloxacin, cephalexin, chloramphenicol, tetracycline,oxytetracycline, vancomycin, cefotetan, moxifloxacin and rifampin (FIG.39).

Minimum inhibitory concentration (MIC) and minimum bactericidalconcentration (MBC) testing was undertaken using CLSI methodology forNCL812 and included ampicillin as a control. Anti-staphylococcalcompounds were then tested against all 23 isolates and minimuminhibitory concentrations (MIC) were determined according to standardprotocols. After the MICs were determined, the minimum bactericidalconcentrations were performed to determine if these compounds arebacteriostatic or bacteriocidal.

Results

The mecA gene was present in 13 isolates of MRSP and negative in 10MSSP. All MRSP isolates were resistant to oxacillin based on discdiffusion (<=17 mm) and E-test MIC (>=0.5 mg/L).

When cefoxitin resistance breakpoint was set at <=24 mm, 3/13 (23%) and5/13 (38%) of MRSP tested were susceptible to cefoxitin. When cefoxitinresistance break point was set at <=30 mm, only 1/13 (7.7%) of MRSPtested at Veterinary Diagnostic Laboratory was susceptible.

The MRSP isolates were resistant to multiple antibiotic classes. Of the13 MRSP isolates, all 13 were susceptible to rifampin. 3/13 (23%) weresusceptible to chloramphenicol; 10/13 (77%) were susceptible tovancomycin.

Interestingly, 3/13 (23%) of the MRSP isolates were susceptible toamoxicillin; 8/13 (62%) were susceptible to cephalothin; 12/13 (92%)susceptible to cefotetan and 12/13 (92%) susceptible to moxifloxacin,

All 23 isolates were susceptible to NCL812 based on MICs. In addition,NCL812 has been shown to be bactericidal based on minimal bactericidalconcentrations (MBC).

The MIC range of NCL812 against the Staphylococcus pseudintermediusisolates was found to be between 1 μg/mL and 4 μg/mL.

TABLE 12 MIC range of NCL812 against Staphylococcus pseudintermediusisolates Isolate AMP NCL812 S1P1 128 4 S2P2 128 2 S3P3 128 2 S4P4 128 1S5P5 16 2 S6P6 64 2 S7P7 128 2 S8P8 128 2 S9P9 32 2 S10P10 64 2 S11P11128 4 S12P12 32 2 S13P13 0.25 2 S14P14 1 2 S15P15 4 4 S16P16 0.25 2S17P17 1 2 S18P18 4 4 S19P19 0.5 4 S20P20 4 4 S21P21 0.1 2 S22P22 8 4S23P23 32 2

The MIC 50 and MIC 90 of NCL812 against the Staphylococcuspseudintermedius isolates was found to be 2 μg/mL and 4 μg/mLrespectively. The MIC mode and MIC range of NCL812 against theStaphylococcus pseudintermedius isolates was found to be 2 μg/mL and 1-4μg/mL respectively.

TABLE 13 Combined MIC values of NCL812 against Staphylococcuspseudintermedius isolates Column1 AMP NCL812 MIC50 (μg/ml) 32 2 MIC90(μg/ml) 128 4 MIC mode 128 2 (μg/ml) MIC range 0.1-128 1-4 (μg/ml)

Methicillin resistant Staphylococcus pseudintermedius (MRSP) is anemerging problem in dogs, cats and horses. Two major clonal MRSPlineages have been reported from dogs in Europe (ST 71) and NorthAmerica (ST 68). There have also been reports of MRSP affecting dogs inJapan and a single case of MRSP in a veterinary worker in Hong Kong.

In this study, MRSP isolates were determined using a combination ofpresence of mecA gene and in vitro resistance to oxacillin. Cefoxitinsusceptibility has been used as a substitute for oxacillin formethicillin resistant Staphylococcus aureus. However, cefoxitin diskdiffusion tests using interpretive guidelines recommended for humanisolates of methicillin resistant Staphylococcus aureus and coagulasenegative staphylococci are unreliable in identifying MRSP. A cefoxitinbreakpoint resistance of <=30 mm=resistant and >=31=susceptible has beenproposed by Bemis et al, 2012. This study is in agreement that thisbreakpoint may be more reliable in predicting methicillin resistantStaphylococcus pseudintermedius. MRSP isolates are generally resistantto multiple antibiotic classes. Bacterial culture and antibioticsusceptibilities are therefore recommended for all suspect MRSPinfections to allow appropriate selection of antibiotics. A limitationnoted in this study is the apparent in vitro susceptibility of MRSPisolates to amoxicillin and cephalosporins (cephalothin and cefotetan).NCL812 was effective against all 23 isolates of both MSSP and MRSP. Alarger scale study is warranted to confirm the effectiveness of NCL812against Staphylococcus pseudintermedius as it may provide a safealternative antibiotic option for emerging MRSP infections in domesticanimals.

Example 7

Activity of NCL812 against gram-negative organisms.

The aim of this study was to determine if a target of the antibacterialactivity of NCL812 is present within gram-negative cells. To determineif a target of NCL812 is within the gram-negative cell, the outermembrane, and most of the cell wall, was removed using ampicillin, thenthe modified cells (known as spheroplasts) were treated with NCL812 atvarious concentrations.

Induction of Spheroplast State

E. coli ATCC 25922 was grown overnight on agar at 37° C. Two coloniesfrom the overnight incubation were used to inoculate ˜20 ml of cationadjusted mueller hinton broth. Inoculated broth was incubated for 18hours at 37° C. Six ml of overnight broth culture was added to 20 ml ofsupplemented cation adjusted mueller hinton broth (supplemented with 50mg/ml ampicillin, 0.4 M sucrose, 8 mM MgSO₄) and incubated overnight at37° C. Formation of spheroplasts was checked using a phase contrastmicroscope.

Activity of NCL812

Three ml of spheroplast culture was added to each test tube and 50 μl ofDMSO containing the appropriate concentration of NCL812 was added toeach test tube. Fifty microlitres of DMSO only was added to the controltest tube. Spheroplasts were incubated for 24 hours with 20 μl samplestaken at 0, 2, 4, 6, 8 and 24 hours. Twenty microliter samples wereserially diluted 1:10 and 10 ul samples of appropriate dilutions werespotted in triplicate onto brain heart infusion agar. Brain heartinfusion agar was incubated at 37° C. for 48 hours and colonies werecounted at 24 and 48 hours to determine the number of colony formingunits.

Imaging of NCL812 Treated Spheroplasts

Samples were taken after 24 hours of exposure to the NCL812 compound,stained with Trypan blue and imaged.

Results

A spheroplast induction rate above 99% was consistently observed. Atarget of NCL812 was found to be present within E. coli cells with asignificant decrease in the number of colony forming units observed overtime as the concentration of NCL812 increased ≧32 μg/ml (FIG. 40). Theexperiment was repeated in triplicate with one representative shown inFIG. 40. Images of the spheroplasts taken after 24 hours of exposure tothe NCL812 compound showed the development of pleomorphic cellsincreasing in frequency as the concentration of NCL812 increased (datanot presented). These results show the effectiveness of NCL812 as anantibacterial agent against gram-negative bacteria.

Example 8

Formulations of NCL812.

The following formulations were prepared using standard methods in theart.

Formulation A—Topical Formulation—PEG-Based Gel with NCL812

4.0 g PEG 4000;

3.5 g PEG 200;

0.6 propylene glycol;

1.9 g water; and

0.204 g of NCL812.

PEG 4000, PEG 200 and propylene glycol were mixed and heated to 150° C.and until all solid crystals were dissolved. NCL812 was added to waterand sonicated for 30 minutes until fully suspended. The NCL812 solutionand gel solutions were mixed and allowed to cool and solidify.Formulation A demonstrated acceptable viscosity, ease of skinapplication, uniform suspension and consistent and fine texture.

Formulation B—Topical Formulation—PEG-Based Gel with NCL812

3.0 g PEG 4000;

1.0 g PEG 8000;

3.0 g PEG 200;

1.0 g propylene glycol;

1.9 g water; and

0.202 g of NCL812.

PEG 4000, PEG 8000, PEG 200 and propylene glycol were mixed and heatedto 150° C. and until all solid crystals were dissolved. NCL812 was addedto water and sonicated for 30 minutes until fully suspended. The NCL812solution and gel solutions were mixed and allowed to cool and solidify.Formulation B demonstrated acceptable viscosity, ease of skinapplication, uniform suspension and consistent and fine texture.

Formulation C—Topical Formulation—PEG-Based Gel with NCL812-Soluplus

2.5 g PEG 4000;

4.0 g PEG 200;

2.5 g propylene glycol;

1.0 g water; and

1.8 g solid dispersion of NCL812-SoluPlus.

Soluplus was purchased from BASF (www.soluplus.com). NCL812-SoluPlus wasprepared using standard methods in the art.

PEG 4000, PEG 200, NCL812-SoluPlus and propylene glycol were mixed andheated to 150° C. and until all solid crystals were dissolve. Water wasadded and then the solution was sonicated. The solution was allowed tocool and solidify. Formulation C demonstrated acceptable viscosity, easeof skin application, uniform suspension and consistent and fine texture.

Formulation D—Tablet Formulation

30 mg Calcium hydrogen phosphate dehydrate;

80 mg Microcrystalline cellulose;

50 mg Lactose;

8 mg Hydroxypropyl methyl cellulose

1.5 mg Talc

10 mg of NCL812

The excipients were weighed and mixed for 5 minutes. The mixture was fedinto a feed hopper of a tablet press machine and the machine wasoperated according to standard procedures in the art.

Formulation D demonstrated acceptable tablet hardness, disintegrationand frability.

Formulation E—Oral Suspension

2.0 ml Glycerol;

1.5 ml Absolute ethanol;

600 mg NCL812; and

To 60 ml Vehicle (Ora Sweet and Ora Plus, 1:1).

NCL 812 powder was sieved through a 75 μm sieve. 600 mg of sieved NCL812 was mixed with 2.0 ml glycerol and 1.5 ml absolute ethanol. Themixture was placed in a mortar and manually milled until all NCL 812 wassuspended uniformly. The suspension was sonicated for 30 minutes.Vehicle (55 ml of Ora Sweet and Ora Plus mixture) was then added to thesuspension and milled for another 10 minutes. Volume was made up withthe Ora plus and Ora sweet mixture to 60 ml by transferring to ameasuring cylinder

Formulation E demonstrated acceptable suspension and demonstratedacceptable short term stability.

Formulation F—Intramuscular Injection

20 mg/ml Polyvinylpyrrolidone K30 (PVPK30);

0.09 mg/ml NCL812; and

50 ml water.

Two percent of w/v PVP K30 solution was prepared by the addition of 1.0g of PVP K30 to 50 ml of MilliQ water. The solution was then placed in asonicator for 30 minutes to equilibrate and 4.5 mg of NCL 812 was addedto the PVP solution and placed on an incubator shaker at a maximum speedof 10 rpm over a period of 24 hours, with controlled temperature of25±1° C. Solution was transferred to 5 ml vials and checked for clarity,appearance, pH and short-term stability. The pH of solution was 7.25.

Formulation F demonstrated acceptable transparency and short termstability.

Example 9

Release of NCL812 from Formulation B.

The objective of this study was to measure the release of NCL812 fromthe Formulation B prepared in example

Franz diffusion cells were utilized to quantify the release rate of NCL812 from its topical formulations. Five millilitres of absolute ethanol,which was chosen as the desired release medium, was loaded into thereceptor chamber. Temperature of the receptor fluid was kept constant,at 32±1° C. using a water jacket. Acetyl cellulose membranes, with poresize of 0.45 um (Pall Corporation) was selected and placed between donorand receptor chamber. Followed by that, a number of test samples(Formulation B) were loaded into the donor chamber. One millilitre ofreceptor fluid was collected at regular time intervals of 0.25, 0.50,0.75, 1, 2, 3, 4, 5, 6, 7, 8 and 24 hours through the sampling port. Onemillilitre of fresh absolute ethanol was immediately returned to thereceptor chamber. UV-HPLC was utilized to analyse the content of thereceptor fluids attained.

FIG. 41 presents the cumulative release of NCL812 over time. This studydemonstrates that Formulation B provides an acceptable release profilefor NCL812.

Example 10

Synergy studies with other classes of antimicrobial agent.

Methods

The checkerboard method (Gunics et al., 2000 Int. J. Antimicrob. Agents.14:239-42) was used to find interactions (synergy, antagonism, noeffect) of NCL812 in combination with tetracycline, chloramphenicol,erythromycin (macrolide), ampicillin (β-lactam broad-spectrum),gentamicin (aminoglycoside), ciprofloxacin (fluoroquinolone),sulfamethoxazole (sulphonamide), or penicillin G (β-lactamnarrow-spectrum). For initial experiments, a laboratory strain ofStaphylococcus aureus T3-129 was used, however this strain gaveinconsistent results for some of the antimicrobials and a new strain ofStaphylococcus spp. designated MK1 (definitive species identificationcurrently in progress) that was sensitive to all tested antimicrobialswas used in subsequent tests.

Firstly, the MIC of each antibiotic alone was determined in accordanceto CLSI standard guidelines. Secondly, the combination of NCL812 witheach of above antibiotics was tested in duplicate. To evaluate theeffect of the combination the fractional inhibitory concentration (FIC)was calculated for each antibiotic as follows: FIC of testedantibiotic=MIC of tested antibiotic in combination/MIC of antibioticalone; FIC of NCL812=MIC of NCL812 in combination/MIC of NCL812 alone;and FIC_(I)=FIC index=FIC of NCL812+FIC of each tested antibiotic.

According to the checkerboard guidelines, Synergy (S) was defined as anFIC_(I)<0.5. No effect (NE) was defined as 0.5<FIC_(I)<4. Antagonism (A)was defined as a 4<FIC_(I).

Results

MICs, FICs, FIC_(I) and the interaction between NCL812 and eightantibiotics is shown in Table 14. None of the eight tested compounds,representing distinct classes of antimicrobial agent showed eitherpositive (synergism) or negative (antagonism) interaction with NCL812consistent with an additive effect when antibacterial agents are addedto NCL812.

TABLE 14 MICs, FICs, FIC_(I) and the interaction between NCL812 andeight antibiotics. Antibiotic NCL812 MIC (μg/ml) MIC (μg/ml) AntibioticName Experiment Repeat With NCL812 Alone FIC₁ With Antibiotic Alone FIC₂FIC₁ Result Tetracycline¹ 1 1^(st) 0.25 0.5 0.5 1 4 0.25 0.75 NE 2^(nd)0.25 0.5 0.5 1 8 0.125 0.62 NE 2 1^(st) 0.031 0.25 0.125 4 8 0.5 0.625NE 2^(nd) 0.031 0.25 0.125 4 8 0.5 0.625 NE Chloramphenicol¹ 1 1^(st) 48 0.5 1 4 0.25 0.75 NE 2^(nd) 2 4 0.5 2 8 0.25 0.75 NE 2 1^(st) 4 8 0.52 8 0.25 0.75 NE 2^(nd) 0.5 8 0.0625 4 8 0.5 0.562 NE Erythromycin¹ 11^(st) 0.031 0.125 0.25 2 4 0.5 0.75 NE 2^(nd) 0.007 0.125 0.063 2 4 0.50.562 NE 2 1^(st) 0.007 0.25 0.25 2 8 0.25 0.5 NE 2^(nd) 0.007 0.250.031 4 8 0.5 0.531 NE Ampicillin¹ 1 1^(st) 0.125 0.25 0.5 1 4 0.25 0.75NE 2^(nd) 0.25 0.5 0.5 0.125 4 0.031 0.53 NE 2 1^(st) 0.062 0.125 0.5 28 0.25 0.75 NE 2^(nd) 0.125 0.25 0.5 2 8 0.25 0.75 NE Gentamicin² 11^(st) 0.062 0.125 0.5 0.5 4 0.125 0.625 NE 2^(nd) 0.062 0.125 0.5 1 40.25 0.75 NE 2 1^(st) 0.5 1 0.5 1 4 0.25 0.75 NE 2^(nd) 0.007 0.5 0.01562 4 0.5 0.515 NE Ciprofloxacin² 1 1^(st) 0.062 0.125 0.5 2 4 0.5 0.75 NE2^(nd) 0.003 0.125 0.025 4 2 0.5 0.525 NE 2 1^(st) 0.125 0.25 0.5 0.5 40.125 0.625 NE 2^(nd) 0.125 0.25 0.5 0.25 4 0.0625 0.562 NESulfamethoxazole² 1 1^(st) 4 8 0.5 1 4 0.25 0.75 NE 2^(nd) 4 8 0.5 2 40.5 1 NE 2 1^(st) 4 8 0.5 1 4 0.25 0.75 NE 2^(nd) 4 8 0.5 2 4 0.5 1 NEPenicillin G² 1 1^(st) 0.062 0.125 0.5 2 4 0.5 1 NE 2^(nd) 0.062 0.1250.5 2 4 0.5 1 NE 2 1^(st) 0.062 0.125 0.5 2 4 0.5 1 NE 2^(nd) 0.031 0.250.125 2 4 0.5 0.625 NE ¹ S. aureus strain T3-29 ² Staphylococcus spp.Strain MK1 FIC₁ = MIC of anitbiotic in combination with NCL812/MIC ofantibiotic alone FIC₂ = MIC of NCL812 in combination with antibiotic/MICof NCL812 alone FIC_(I) = FIC index

Example 11

The Effects of NCL812 on antimicrobial sensitive isolates ofStaphylococcus aureus and Enterococcus faecalis

Materials and Methods Strain Information

Two Staphylococcus aureus isolates were used in the followingexperiments; S. aureus MK01 a human skin strain, and S. aureus KC01 anequine skin strain. These isolates were identified by Gram stain andbiochemical methods, including the Remel Staphaurex commercial kit. OneEnterococcus faecalis isolate (USA01), was not identified as a VREstrain. As this isolate has previously been speciated, it was notsubjected to further testing, except for observation of pure,characteristic growth on blood agar.

Investigation of Minimum Bactericidal Concentration (MBC) CLSIMethodology

As in previous experiments, 10 μL of the contents of each well startingat the MIC was inoculated on to a Columbia SBA plate and incubated at37° C. for 48 h. Plates were examined at 24 and 48 h and the MBC wasrecorded as the lowest concentration of NCL812 at which no colonies ofbacteria were observed on the plate (or significant inhibition of growthwas observed compared to the control) (CLSI 2005).

Kill Kinetics Assays for S. aureus KC01 & E. faecalis USA01 Method

S. aureus KC01 and E. faecalis USA01, not determined to be MRSA or VRE,respectively, were grown overnight on Columbia SBA at 37° C. A fewcolonies of bacteria were then suspended in CAMHB (cation-adjustedMueller Hinton broth) and adjusted to OD₆₀₀ of 0.08 to 0.10. Thebacterial suspension was diluted 1:10. One millilitre of the bacteriawere added to 9 mL of CAMHB containing various concentrations (up to4×MIC) of NCL, to achieve a final bacterial concentration of 1 to 3×106CFU/mL. The tubes were incubated at 37° C., with constant shaking. Inorder to determine the number of viable bacteria present at various timepoints, a 100 μL aliquot was removed from each tube and diluted. Then,100 μL of each dilution were spread onto colony count agar, induplicate, and incubated for 48 h at 37° C. After 24 h the numbers ofcolonies present on each plate were counted and therefore the number ofviable bacteria present in the original suspension enumerated. Plateswere re-checked after 48 hours.

Results Minimum Inhibitory Concentration (MIC)

The NCL812 MIC for isolates S. aureus MK01 and KC01, and E. faecalisUSA01 was investigated. The results were: S. aureus MK01=4-8 μg/mL, S.aureus KC01=2 μg/mL, E. faecalis USA 01=4 μg/mL.

S. aureus isolates MK01 and KC01 were investigated and no growth, orgrowth only at low concentrations of NCL812 (2 μg/ml), was observed,indicating that NCL812 is bactericidal against S. aureus. For the E.faecalis isolate tested (USA01) however, growth of bacteria was observedat all concentrations of NCL812 tested. There was an obvious reductionin the number of bacteria with increasing concentration, but growth waspresent compared with no growth for S. aureus. A summary of theseresults can be seen in Table 15. Table 15 shows the results for NCL812MBC tests on two non-MRSA S. aureus isolates and one non-VRE E. faecalisisolate. Each MBC test was performed in duplicate. No change in theresults was observed at 48 h. Table 16 shows NCL812 MBC values (μg/mL)for 20 MRSA isolates. Each MBC test was performed in duplicate startingfrom NCL812 MIC concentration to 16 times of MIC. Table 17 shows NCL812MBC values (μg/ml) for 10 VRE isolates. Each MBC test was performed induplicate starting from NCL812 MIC concentration to 32 times the MIC.

TABLE 15 NCL812 MBC tests on two non-MRSA Staphylococcus aureus isolatesand one non VRE Enterococcus faecalis isolate. NCL812 MBC Organism/ 2 48 16 32 64 128 Sample No. μg/ml μg/ml μg/ml μg/ml μg/ml μg/ml μg/ml S.aureus 1^(st) + 0 0 0 0 0 0 (KC01) 2^(nd) + + + 0 0 0 0 S. aureus 1^(st)0(5) 0 0 0(N) 0(N) 0(N) 0(N) (MK01) 2^(nd) 0 0 0 0 0 0 0 E. faecalis1^(st) N + (488) + + + (7) + (1) + (USA01) 2^(nd) N + + + + + + + =Growth on Sheep Blood Agar; 0 = No Growth on Sheep Blood Agar; N = NotCultured; Numbers in Parenthesis are the Number of Bacteria Growingafter 24 hours per ml of sample (CFU/ml)

TABLE 16 NCL812 MBC values (μg/ml) for 20 MRSA isolates. NCL812 MBC 4 816 32 64 Organism/Sample No. μg/ml μg/ml μg/ml μg/ml μg/ml MRSA 1 1^(st)0 0 0 0  N** 2^(nd) 0 0 0 0 N MRSA 2 1^(st) 0 0 0 0 N 2^(nd) 0 0 0 0 NMRSA 3 1^(st) 0  GB* 0 0 N 2^(nd) 0 0 0 0 N MRSA 4 1^(st) 0 0 0 0 N2^(nd) 0 0 0 0 N MRSA 5 1^(st) 0 0 0 0 N 2^(nd) 0 0 0 0 N MRSA 6 1^(st)0 0 0 0 N 2^(nd) 0 0 0 0 N MRSA 7 1^(st) 0 0 0 0 N 2^(nd) 0 GB 0 0 NMRSA 8 1^(st) GB 0 0 0 N 2^(nd) 0 0 0 0 N MRSA 9 1^(st) 0 0 0 0 N 2^(nd)0 0 0 0 N MRSA 10 1^(st) 0 0 0 0 N 2^(nd) 0 0 0 0 N MRSA 11 1^(st) 0 0 00 N 2^(nd) GB 0 0 0 N MRSA 12 1^(st) 0 0 0 0 N 2^(nd) 0 GB 0 0 N MRSA 131^(st) 0 0 0 0 N 2^(nd) 0 0 0 0 N MRSA 14 1^(st) 0 0 0 0 N 2^(nd) 0 0 00 N MRSA 516 1^(st) 0 0 0 0 0 2^(nd) 0 0 0 0 0 MRSA 570 1^(st) 0 0 0 0 02^(nd) 0 0 0 0 0 MRSA 580 1^(st) 0 0 0 0 0 2^(nd) 0 0 0 0 0 MRSA 6061^(st) 0 0 0 0 GB 2^(nd) 0 0 0 0 0 MRSA 610 1^(st) 0 0 0 0 0 2^(nd) 0 GB0 0 0 GB = Bacterial Growth on Sheep Blood Agar N** = Not cultured onSheep Blood Agar

TABLE 17 NCL812 MBC values (μg/ml) for 10 VRE isolates. NCL812 MBCOrganism/ 2 4 8 16 32 64 128 Sample No. μg/ml μg/ml μg/ml μg/ml μg/mlμg/ml μg/ml VRE 26c(dc) 1^(st) 90* 20 4000 M M M M 2^(nd) 0 70 3500 M MM M VRE 37c 1^(st) 500  100 20 250 M M M 2^(nd)  M 50 100 1100 1400 M MVRE 35t 1^(st) 0 0 0 720 0 0 0 2^(nd) 0 0 0 0 10 20 10 VRE 16c(dc)1^(st) 90  330 0 M M M M 2^(nd) 200  0 20 M M M M VRE 23c 1^(st) 0 12020 10 M M M 2^(nd) 0 0 0 0 570 M M VRE 25c 1^(st) 0 0 M M M M M 2^(nd)20  20 M M M M M VRE 16c 1^(st) 10  820 980 M M M M 2^(nd)  M 790 890 MM M M VRE 19t 1^(st) 0 0 0 180 10 110 M 2^(nd) 30  0 0 70 40 M M VRE 14t1^(st) 10  0 10 0 180 970 M 2^(nd) 0 0 0 40 780 M M VRE 12c 1^(st) 0 0 0M M M M 2^(nd) 0 M 300 M M M M *Number of bacteria growing after 24hours per ml of sample (CFU/ml); M = many bacteria growing on the plate(too many to count)Kill Kinetics Assays for S. aureus KC01 & E. faecalis USA01 Method

Colony counts were performed at t=0, 120, 240, and 360 min, then againat 24 h. At the 2 h time point S. aureus KC01 showed a minimum of a 2.5log₁₀ reduction in bacterial numbers from initial numbers, and greaterthan a 3 log₁₀ reduction in comparison to the control at the same timepoint. A minimum of a 2 log₁₀ reduction was still evident at 6 hincubation, however after 24 h the numbers of bacteria present hadincreased and this was not significantly different to the control.

Similar results were obtained with E. faecalis USA01, however thereduction in bacterial numbers observed was less than for S. aureusKC01. A 2 log₁₀ reduction in CFU/mL was observed at 2 h, compared to thegrowth control. However, the reduction in CFU/mL compared to theoriginal bacterial numbers was only just greater than 1 log₁₀. Atconcentrations of 4-16 μg/mL of NCL812 this reduction in bacterialnumbers remained consistent until the 6 h time point. At concentrationsof 32 and 64 μg/mL however, there was approximately a 1 log₁₀ rise inbacterial numbers over the same time period. At 24 h bacterial numbersat all concentrations had increased to almost the same level as thegrowth control.

The results observed with these strains of S. aureus and E. faecalis areconsistent with the results observed for the kill kinetics assay for allMRSA and VRE isolates tested. The kill kinetics assay of Staphylococcusaureus KC01 at different concentrations of NCL812, up to 24 h incubationare shown in FIG. 42. The kill kinetics assay of Enterococcus faecalisUSA01 at different concentrations of NCL812, up to 24 h incubation areshown in FIG. 43.

Example 12

A method of treating bacterial infection in vivo by the administrationof NCL812.

The objective of this study was to determine the efficacy of anInvestigational Veterinary Product containing NCL812 in the treatment ofa skin infection in mice

Summary of the Model

A useful animal model system should be clinically relevant,experimentally robust, ethically acceptable, convenient to perform andshould provide reliable and reproducible results. There are many animalmodels of topical skin infection that have been described including thecroton oil-inflamed skin model (Akiyama, H., H. Kanzaki, Y. Abe, J. Tadaand J. Arata (1994). “Staphylococcus aureus infection on experimentalcroton oil-inflamed skin in mice.” Journal of Dermatological Science8(1): 1-10), the burnt skin model (Stieritz, D. D., A. Bondi, D.McDermott and E. B. Michaels (1982). “A burned mouse model to evaluateanti-pseudomonas activity of topical agents.” Journal of AntimicrobialChemotherapy 9(2): 133-140), the skin suture-wound model (McRipley, R.J. and R. R. Whitney (1976). “Characterization and Quantitation ofExperimental Surgical-Wound Infections Used to Evaluate TopicalAntibacterial Agents.” Antimicrobial Agents and Chemotherapy 10(1):38-44), the skin tape-stripping model (Kugelberg, E., T. NorstrÖm, T. K.Petersen, T. Duvold, D. I. Andersson and D. Hughes (2005).“Establishment of a Superficial Skin Infection Model in Mice by UsingStaphylococcus aureus and Streptococcus pyogenes.” Antimicrobial Agentsand Chemotherapy 49(8): 3435-3441) and the linear full thickness scalpelcut method (Guo, Y., R. I. Ramos, J. S. Cho, N. P. Donegan, A. L. Cheungand L. S. Miller (2013). “In Vivo Bioluminescence Imaging To EvaluateSystemic and Topical Antibiotics against Community-AcquiredMethicillin-Resistant Staphylococcus aureus-Infected Skin Wounds inMice.” Antimicrobial Agents and Chemotherapy 57(2): 855-863).

Preliminary studies prior to the conduct of the current studyestablished a new method of skin infection arising from a detailed studyof the models mentioned above. Briefly, study mice are anaesthetised, apatch of dorsal skin is clipped to reveal the skin and a circular areaof skin is removed with a hand held punch, leaving a wound on the dorsumwith a central cavity. The wound is infected with a known number of thechallenge organism. Approximately four to six hours after infection, thewound is either treated topically with a vehicle formulation or anactive formulation. The infected skin wound is retreated every 12 hoursfor a total of 14 treatments. Mice are humanely euthanased, the area ofthe original infected wound is dissected and removed and its bacterialcontent quantified by standard microbiologic tests. In this way, thechange in bacterial concentration due to treatment with the activeformulation can be readily determined by examining the reduction inbacterial burden compared with the vehicle control.

Materials and Methods Preparation of Infection Inoculum

Fresh cultures of bacteria (Staphylococcus aureus) were grown on SheepBlood Agar at 37° C. for 16-18 hours. A few typical colonies wereselected and suspended in 10 ml of Tryptic Soy Broth and incubatedovernight in a shaking incubator (240 rpm) at 37° C. The overnightsuspension was vortexed and diluted (1:100) in fresh Tryptic soy broth(100 μl [0.1 ml] in 9.9 ml broth). The fresh suspension was incubatedfor 3 hours in a shaking incubator (as above) in order to obtainmid-logarithmic phase bacteria. Bacteria were pelleted throughcentrifugation at 7,500 rpm for 10 mins. Broth supernatant was removedand bacteria suspended in 10 ml Phosphate Buffered Saline (PBS). Thesesteps were repeated a further two times. The density of the suspensionwas checked by measuring absorbance at 600 nm, using a spectrophotometerwith saline as a blank, to confirm the target density at a reading ofapproximately 0.100, consistent with a bacterial density of 2.5×10⁷CFU/ml. The suspension was placed into a rack placed into a lockabletransport box with an ice brick to maintain refrigeration duringtransport, followed by storage in cool room upon arrival at the mouseskin infection laboratory. Final suspension was mixed thoroughly beforeinoculating the skin wounds created in mice.

In order to ensure the purity and accuracy of the suspension, thefollowing steps were performed prior to placement into lock box.

Purity of bacterial suspension ensured by spreading 100 μl of the finalsuspension onto a SBA (sheep blood agar) plate which was incubated at37° C. for 18 hours and examined to confirm uniform growth of one colonytype. Viable counts were performed on final suspension by preppingsaline in Eppendorf tubes (approximately 900 ul per tube), removing 100μl sample and adding to first Eppendorf tube, vortexing the mixture andrepeating using 2^(nd) Eppendorf tube containing saline. This processwas continued for 5-6 tubes. Finally, 100 μl of 5^(th) and 6^(th)dilutions were plated out on plate count agar, incubated at 37° C. for18 hours and colony counts performed to confirm that the CFU/ml wasapproximately 2.5×10⁷. Following inoculation of the wounds, this processwas repeated to ensure that no contamination or decrease in viablecounts had occurred during the time of the surgery.

Skin Wound Surgical Procedure

Each mouse was placed into induction chamber and anaesthesia inducedusing 2% isoflurane. Eyes of each anaesthetised mouse were covered withveterinary eye lubricant in order to prevent corneal dehydration. Eachmouse removed from induction chamber and placed onto surgical area, infront of individual aesthetic nose cone. While under anaesthesia eachmouse was monitored for assessment of depth of anaesthesia (response topain, blink reflex, skeletal muscle tone) and respiratory and cardiacfunction. Back skin hair was shaved from surgical area with mechanicalclippers. Shaved area was cleaned using 70% ethanol applied to papertowel followed by 10% w/v povidone-iodine solution. Once the iodinesolution was dry, a subcutaneous injection of the nonsteroidalanti-inflammatory agent meloxicam was administered. Dorsal skin waspinched gently to allow creation of a circular full-thickness woundusing ear punch/biopsy punch. Vehicle control and NCL812 treated micehad wounds inoculated with 10 μl of bacterial suspension using amicropipette (2.5×10⁵ CFU/10 μl). Once the bacterial suspension was dry,mice were placed into individual recovery boxes labelled with the mousenumber. The time of inoculation was recorded. Initial body weights ofeach mouse were recorded on the appropriate score sheet. Mice recoveredto full consciousness within 5 minutes. Recovered mice were returned toindividual housing and monitored hourly for post-surgical or anaestheticcomplications.

Post-Surgical Care (4 Hours Post-Surgery)

Mice were assessed for post-surgical complications and observations wererecorded on clinic record sheet. Each mouse was carefully removed fromIVC and placed into an assessment container, avoiding excessive handlingor touching of the surgical site. Once the mouse was inside assessmentcontainer, it was assessed and the observations recorded on thepost-surgical clinical record sheet. Whenever the suggested wellnessbreakpoints were reached, post-operative analgesia was administered andrecorded on the clinical record sheet.

Animal Monitoring and Daily Care

Antibiotic Administration (7 am and 6 pm). The first administration ofvehicle or NCL812 ointment occurred 4 hours post-surgically. Eachointment container was weighted prior to administration and the weightrecorded. Each mouse was carefully restrained. Ointment (vehicle orNCL812) was applied to the lesion area and the treated mouse wasreturned to IVC where each mouse was observed to ensure ointment was notimmediately removed by grooming. The weight of the ointment containerpost-administration was recorded. The vehicle and active NCL productswere applied to the skin wound each 12 hours following the firstadministration for a total of 14 consecutive treatments. The NCL812ointment (Formulation B, as presented in Example 8) contained robenidineat a concentration of 20 mg/g. Approximately 0.1-0.2 g of ointment wasapplied on each occasion, delivering a total topical dose of NCL812between 28 and 56 mg to mice weighing between 18 g and 25 g.

Daily Monitoring. Monitoring of each mouse took place once daily ataround 12 pm. Each mouse carefully removed from IVC and placed intoobservation container, avoiding excessive handling or touching surgicalsite. The coat, posture, eyes, behaviour, vocalisation and activitywhilst in the container were carefully assessed and observationsrecorded on assessment sheet. Mouse faeces (either on floor of cage orin container) were checked for consistency and observations recorded.The weight of each mouse was determined whilst it was in the containerand change in body weight calculated and recorded. The observationcontainer was disinfected with ethanol and set aside to dry while afresh container was used for the next mouse. For every second day, micewere again anaesthetised using 2% isoflurane and photographed using aruler for size referencing. These photos were used to assess lesion sizeand infection progression during the trial period.

Tissue Analysis and Assessment of Antibacterial Efficacy

At the end of the 7 day skin wound assessment period, all test mice wereeuthanased prior to wound collection for post mortem examination. Theskin wound was dissected from the dorsum of each mouse. The sample wasplaced in a sample tube and weighed before 1 ml PBS and sterile tissuehomogenisation beads were added. Tissue samples were homogenised for 10mins using a tissue homogeniser (Next Advance Bullet Blender) and thenvortexed for approximately 30 seconds. 100 μl of supernatant was removedand placed into an Eppendorf tube containing 900 μl of PBS. Thisprocedure was repeated using serial dilutions for a total of 8dilutions. Finally, 100 μl of each dilution was pipetted onto a platecount agar in duplicate and incubated overnight at 37° C. Tenmicrolitres of original suspension was placed onto sheep blood agar toassess culture purity and incubated overnight at 37° C. The followingday, viable counts were performed using incubated plate count agarplates and the identity of Staphylococcus aureus (the challengeorganisms) as the harvested strain was confirmed.

Results

The mean colony count per gram of tissue observed in vehicle treatedgroup was 5,888,436 (6.77 log 10). The mean colony count per g of tissueobserved in NCL812 group was 141,254 (5.15 log 10). The log 10 colonyforming units per gram of tissue and % reduction are summarised in thefollowing table.

TABLE 15 Log10 colony forming units per gram of tissue and percentagereduction following topical administration of vehicle and treatmentTreatment Log₁₀(CFU/g) % reduction Vehicle 6.77 NCL812 5.15 97.6It is clear from this table that treatment with NCL812 leads to highlevel reduction in the number of infecting Staphylococcus aureus. Theseresults demonstrate effective treatment of a bacterial colonisation orinfection in vivo.

1. A method of treating or preventing a bacterial colonisation orinfection in a subject, the method comprising the step of: administeringa therapeutically effective amount of robenidine, or a therapeuticallyacceptable salt thereof, to the subject, wherein the bacterialcolonisation or infection is caused by a bacterial agent.
 2. The methodaccording to claim 1, wherein the subject is selected from the groupconsisting of: human, canine, feline, bovine, ovine, caprine, porcine,avian, piscine and equine species.
 3. The method according to claim 1,wherein the robenidine is administered to the subject at a dose in therange of 0.1 mg/kg to 250 mg/kg bodyweight.
 4. The method according toclaim 1, wherein the bacterial agent is gram positive.
 5. The methodaccording to claim 4, wherein the bacterial agent is selected from thegroup consisting of: Abiotrophia defectiva, Acholeplasma spp.,Actinobaculum suis, Actinomyces bovis, Actinomyces europaeus,Actinomyces georgiae, Actinomyces gerencseriae, Actinomycesgraevenitzii, Actinomyces hordeovulneris, Actinomyces israelii serotypeII, Actinomyces israelii, Actinomyces meyeri, Actinomyces naeslundii,Actinomyces neuii, Actinomyces odontolyticus, Actinomyces radingae,Actinomyces spp, Actinomyces turicensis, Actinomyces viscosus,Alloscardovia omnicolens, Anaerococcus hydrogenalis, Anaerococcuslactolyticus, Anaerococcus murdochii, Anaerococcus octavius,Anaerococcus prevotii, Anaerococcus tetradius, Anaerococcus vaginalis,Arcanobacterium (Actinomyces) bernardiae, Arcanobacterium (Actinomyces)pyogenes, Arcanobacterium bernardiae, Arcanobacterium cardiffensis,Arcanobacterium funkei, Arcanobacterium haemolyticum, Arcanobacteriumhoustonensis, Arcanobacterium lingnae, Arcanobacterium pyogenes(Actinomyces pyogenes), Arthrobacter, Atopobium minutum, Atopobiumparvulum, Atopobium rimae, Atopobium spp, Atopobium vaginae, Bacillusanthracis, Bacillus cereus, Bacillus circulans, Bacillus licheniformis,Bacillus megaterium, Bacillus melaninogenicus, Bacillus pumilus,Bacillus sphaericus, Bacillus subtilis, beta haemolytic Steptococcusspp, Bifidobacteria adolescentis, Bifidobacteria dentium, Bifidobacteriascardovii, Bifidobacteria, Brevibacillus brevis, Brevibacilluslaterosporus, Brevibacterium, Bulleidia extructa, Catabacterhongkongensis, CDC coryneform groups F-1 and G, Clostridiium tetani,Clostridium baratii, Clostridium bifermentans, Clostridium botulinum(types A, B, C, D, E, F, G), Clostridium botulinum, Clostridiumbutyricum, Clostridium chauvoei, Clostridium colinum, Clostridiumdifficile, Clostridium haemolyticum, Clostridium histolyticum,Clostridium novyi type A, Clostridium novyi type B, Clostridium novyi,Clostridium perfringens type A, Clostridium perfringens types A-E,Clostridium perfringens, Clostridium piliforme, Clostridium ramosum,Clostridium septicum, Clostridium sordelli, Clostridium sphenoides,Clostridium spiroforme, Clostridium spp, Clostridium tertium,Clostridium tetani, Collinsella aerofaciens, Corynebacterium accolens,Corynebacterium afermentans afermentans, Corynebacterium afermentanslipophilum, Corynebacterium amycolatum, Corynebacterium argentoratense,Corynebacterium aurimucosum, Corynebacterium auris, Corynebacteriumbovis, Corynebacterium confusum, Corynebacterium cystidis,Corynebacterium diphtheria, Corynebacterium freneyi, Corynebacteriumglucuronolyticum, Corynebacterium jeikeium, Corynebacteriumkroppenstedtii, Corynebacterium kutscheri, Corynebacteriumlipophiloflavum, Corynebacterium macginleyi, Corynebacteriummatruchotii, Corynebacterium minutissimum, Corynebacterium pilosum,Corynebacterium propinquum, Corynebacterium pseudodiphtheriticum,Corynebacterium pseudotuberculosis, Corynebacterium renale,Corynebacterium riegelii, Corynebacterium simulans, Corynebacteriumstriatum, Corynebacterium sundvallense, Corynebacterium thomssensii,Corynebacterium tuberculostearum, Corynebacterium ulcerans,Corynebacterium urealyticum, Corynebacterium xerosis, Crossiella equi,Dermabacter, Dermatophilus congolense, Dermatophilus congolensis,Eggerthella brachy, Eggerthella hongkongensis, Eggerthella infirmum,Eggerthella lenta, Eggerthella minutum, Eggerthella nodatum, Eggerthellasaphenum, Eggerthella sinensis, Eggerthella sulci, Eggerthella tenue,Eggerthella, Enterococcus avium, Enterococcus bovis, Enterococcuscasseliflavus/flavescens, Enterococcus cecorum, Enterococcus dispar,Enterococcus durans, Enterococcus faecalis, Enterococcus faecium,Enterococcus gallinarum, Enterococcus gilvus, Enterococcus hirae,Enterococcus italicus, Enterococcus malodoratus, Enterococcus mundtii,Enterococcus pallens, Enterococcus pseudoavium, Enterococcus raffinosus,Enterococcus sanguinicola, Enterococcus spp, Erysipelothrixrhusiopathiae, Eubacterium, Filifactor alocis, Finegoldia magna,Gallicola barnesae, Gemella asaccharolytica, Gemella bergeri, Gemellacuniculi, Gemella haemolysans, Gemella morbillorum, Gemella palaticanis,Gemella sanguinis, Gordonia spp., Granulicatella adiacens,Granulicatella elegans, Granulicatella para-adiacens, Kytococcusschroeteri, Lactobacillus acidophilus, Lactobacillus casei,Lactobacillus fermentum, Lactobacillus gasseri, Lactobacillus iners,Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus species,Lactobacillus ultunensis, Leifsonia aquatic, Leuconostoc citreum,Leuconostoc lactis, Leuconostoc mesenteroides, Leuconostocparamesenteroides, Leuconostoc pseudomesenteroides, Listeria grayi,Listeria innocua, Listeria ivanovii, Listeria monocytogenes, Listeriaseeligeri, Listeria welshimeri, Microbacterium, Mobiluncus curtisii,Mobiluncus mulieris, Mobiluncus spp, Mogibacterium timidum,Mogibacterium vescum, Moryella indoligenes, Mycobacterium senegalense,Mycobacterium abscessus, Mycobacterium africanum, Mycobacteriumarupense, Mycobacterium asiaticum, Mycobacterium aubagnense,Mycobacterium avium complex, Mycobacterium avium subsp paratuberculosis,Mycobacterium avium, Mycobacterium bolletii, Mycobacterium bovis,Mycobacterium branderi, Mycobacterium canettii, Mycobacterium caprae,Mycobacterium celatum, Mycobacterium chelonae, Mycobacterium chimaera,Mycobacterium colombiense, Mycobacterium conceptionense, Mycobacteriumconspicuum, Mycobacterium elephantis, Mycobacterium farcinogenes,Mycobacterium florentinum, Mycobacterium fortuitum group, Mycobacteriumgenavense, Mycobacterium goodii, Mycobacterium haemophilum,Mycobacterium heckeshornense, Mycobacterium heidelbergense,Mycobacterium houstonense, Mycobacterium immunogenum, Mycobacteriuminterjectum, Mycobacterium intracellulare, Mycobacterium kansasii,Mycobacterium lacus, Mycobacterium lentiflavum, Mycobacterium leprae,Mycobacterium lepraemurium, Mycobacterium mageritense, Mycobacteriummalmoense, Mycobacterium marinum, Mycobacterium massiliense,Mycobacterium microti, Mycobacterium montefiorense (eels), Mycobacteriummoracense, Mycobacterium mucogenicum, Mycobacterium nebraskense,Mycobacterium neoaurum, Mycobacterium novocastrense, Mycobacteriumpalustre, Mycobacterium paratuberculosis (Johne's Disease),Mycobacterium parmense, Mycobacterium phlei, Mycobacterium phocaicum,Mycobacterium pinnipedii, Mycobacterium porcinum, Mycobacteriumpseudoshottsii (fish), Mycobacterium pseudotuberculosis, Mycobacteriumsaskatchewanense, Mycobacterium scrofulaceum, Mycobacterium senuense,Mycobacterium septicum, Mycobacterium simiae, Mycobacterium smegmatis,Mycobacterium spp, Mycobacterium szulgai, Mycobacteriumterrae/chromogenicum complex, Mycobacterium triplex, Mycobacteriumtuberculosis, Mycobacterium tusciae, Mycobacterium ulcerans,Mycobacterium wolinskyi, Mycobacterium xenopi, Mycobacterium, Nocardiaasteroides, Nocardia brasiliensis, Nocardia farcinica, Nocardia nova,Nocardia otitidiscaviarum, Nocardia spp, Nocardia transvalensis,Oerskovia, Olsenella oral spp, Olsenella profuse, Olsenella uli,Oribacterium sinus, Paenibacillus alvei, Parvimonas micra, Pediococcus,Peptococcus indolicus, Peptococcus niger, Peptoniphilusasaccharolyticus, Peptoniphilus gorbachii, Peptoniphilus harei,Peptoniphilus indolicus, Peptoniphilus ivorii, Peptoniphilus lacrimalis,Peptoniphilus olsenii, Peptostreptococcus anaerobius, Peptostreptococcusstomatis, Propionibacterium acnes, Propionibacterium granulosum,Propionibacterium propionicum, Propionibacterium, Pseudoramibacteralactolyticus, Rhodococcus equi, Rhodococcus erythropolis, Rhodococcusfasciens, Rhodococcus rhodochrous, Rothia, Ruminococcus productus,Slackia exigua, Slackia heliotrinireducens, Solobacterium moorei,Staphylococcus arlettae, Staphylococcus aureus subsp. anaerobius,Staphylococcus aureus, Staphylococcus auricularis, Staphylococcuscapitis subsp. capitis, Staphylococcus capitis subsp. urealyticus,Staphylococcus capitis, Staphylococcus caprae, Staphylococcus carnosus,Staphylococcus caseolyticus, Staphylococcus chromogenes, Staphylococcuscohnii subsp. cohnii, Staphylococcus cohnii subsp. urealyticus,Staphylococcus cohnii, Staphylococcus condimenti, Staphylococcusdelphini, Staphylococcus epidermidis, Staphylococcus equorum,Staphylococcus felis, Staphylococcus fleurettii, Staphylococcusgallinarum, Staphylococcus haemolyticus, Staphylococcus hominis,Staphylococcus hyicus, Staphylococcus intermedius, Staphylococcuskloosii, Staphylococcus lentus, Staphylococcus lugdunensis,Staphylococcus lutrae, Staphylococcus muscae, Staphylococcus nepalensis,Staphylococcus pasteuri, Staphylococcus pettenkoferi, Staphylococcuspiscifermentans, Staphylococcus pseudintermedius, Staphylococcuspulvereri, Staphylococcus saccharolyticus, Staphylococcus saprophyticus,Staphylococcus schleiferi subsp. coagulans, Staphylococcus schleiferi,Staphylococcus sciuri, Staphylococcus simiae, Staphylococcus simulans,Staphylococcus spp, Staphylococcus succinus, Staphylococcus vitulinus,Staphylococcus warneri, Staphylococcus xylosus, Staphylococcusvitulinus, Stomatococcus mucilaginosus (reclassified as Rothiamucilaginosa), Streptococcus agalactiae, Streptococcus anginosus speciesgroup (Streptococcus intermedius, Streptococcus constellatus, andStreptococcus anginosus), Streptococcus bovis species group (S.gallolyticus subsp. gallolyticus (formerly S. bovis biotype I),Streptococcus bovis, Streptococcus canis, Streptococcus dysgalactiaesubsp. dysgalactiae, S. equi subsp. equi, S. equi subsp. zooepidemicus,S. porcinus, S. canis, S. suis, S. iniae), Streptococcus dysgalactiaesubsp. equisimilis, Streptococcus dysgalactiae, Streptococcus equi(Streptococcus equi subsp equi), Streptococcus equi subsp.zooepidemicus, Streptococcus equi, Streptococcus equinus, Streptococcusequisimilis (Streptococcus dysgalactiae subsp equisimilis),Streptococcus gallolyticus subsp. pasteurianus (formerly S. bovisbiotype II/2), Streptococcus infantarius subsp Infantarius,Streptococcus lutetiensis (formerly S. bovis biotype II/1),Streptococcus mitis species group (S. cristatus, S. infantis, S. mitis,S. oxalis, S. peroris, S. orisratti), and Streptococcus mutans speciesgroup (S. cricetus, S. downei, S. ferus, S. hyovaginalis, S. macaccae,S. mutans, S. ratti, S. sobrinus, Sanguinis Group, S. gordonii, S.parasanguinis, S. sanguinis), Streptococcus pneumoniae, Streptococcusporcinus, Streptococcus pyogenes, Streptococcus salivarius species group(S. alactolyticus, S. hyointestinalis, S. infantarius, S. salivarius, S.thermophilus, S. vestibularis), Streptococcus spp, Streptococcus suis,Streptococcus uberis, Streptococcus zooepidemicus (Streptococcus equisubsp zooepidemicus), Streptococcus zooepidemicus, Trueperellaabortisuis, Trueperella bernardiae, Trueperella bialowiezensis,Trueperella bonasi, Trueperella pyogenes (Arcanobacterium pyogenes),Tsukamurella spp., Turicella, and Turicibacter sanguine.
 6. The methodaccording to claim 5, wherein the bacterial agent is selected from thegroup consisting of: Staphylococcus aureus, Staphylococcuspseudintermedius, Streptococcus pneumoniae, Streptococcus pyogenes,Streptococcus agalactiae, Enterococcus faecium, Enterococcus faecalis,and Clostridium difficile.
 7. The method according to claim 1, whereinthe bacterial agent is gram negative. 8-21. (canceled)
 22. Anantibacterial pharmaceutical composition comprising a therapeuticallyeffective amount of robenidine, or a therapeutically acceptable saltthereof, and optionally a pharmaceutically acceptable excipient orcarrier.
 23. An antibacterial veterinary composition comprising atherapeutically effective amount of robenidine, or a therapeuticallyacceptable salt thereof, and optionally a veterinary acceptableexcipient or carrier. 24-33. (canceled)
 34. A medical device when usedin a method of treating or preventing a bacterial colonisation orinfection in the subject, wherein the medical device comprises thecomposition according to claim 22, wherein the composition furthercomprises a further antimicrobial agent selected from the groupconsisting of an antibacterial agent and an antifungal agent. 35.(canceled)
 36. A method of killing bacteria, the method including thestep of contacting the bacteria with robenidine, or a therapeuticallyacceptable salt thereof. 37-38. (canceled)
 39. A medical device whenused in a method of treating or preventing a bacterial colonisation orinfection in the subject, wherein the medical device comprises thecomposition according to claim 23, wherein the composition furthercomprises a further antimicrobial agent selected from the groupconsisting of an antibacterial agent and an antifungal agent.